Humanized antibodies to LIV-1 and use of same to treat cancer

ABSTRACT

The invention provides humanized antibodies that specifically bind to LIV-1. The antibodies are useful for treatment and diagnoses of various cancers as well as detecting LIV-1.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.13/990,778 filed May 30, 2013, which is a U.S. National Stage ofPCT/US2011/063612 filed Dec. 6, 2011, which is a nonprovisional andclaims the benefit of 61/420,291, filed Dec. 6, 2010 and 61/446,990,filed Feb. 25, 2011, each incorporated by reference in its entirety forall purposes.

REFERENCE TO A SEQUENCE LISTING

This application includes an electronic sequence listing in a file named“472222_SEQLST.TXT” created on Apr. 19, 2016, and having a size of101,037 bytes. The information in this file is hereby incorporated byreference.

BACKGROUND

LIV-1 is a member of the LZT (LIV-1-ZIP Zinc Transporters) subfamily ofzinc transporter proteins. Taylor et al., Biochim. Biophys. Acta1611:16-30 (2003). Computer analysis of the LIV-1 protein reveals apotential metalloprotease motif, fitting the consensus sequence for thecatalytic zinc-binding site motif of the zinc metalloprotease. LIV-1mRNA is primarily expressed in breast, prostate, pituitary gland andbrain tissue.

The LIV-1 protein has also been implicated in certain cancerousconditions, e.g. breast cancer and prostate cancer. The detection ofLIV-1 is associated with estrogen receptor-positive breast cancer,McClelland et al., Br. J. Cancer 77:1653-1656 (1998), and the metastaticspread of these cancers to the regional lymph nodes. Manning et al.,Eur. J. Cancer 30A:675-678 (1994).

SUMMARY OF THE CLAIMED INVENTION

The invention further provides a humanized antibody comprising a matureheavy chain variable region having an amino acid sequence at least 90%identical to HB (SEQ ID NO:10) and a mature light chain variable regionat least 90% identical to LB (SEQ ID NO:15). Optionally, the antibodycomprises a mature heavy chain variable region having an amino acidsequence at least 95% identical to HB and a mature light chain variableregion at least 95% identical to LB. Optionally, in any such antibody,positions H29, H30 and H76 are occupied by I, E and N, and L36 isoccupied by Y. Optionally, any difference in the variable regionframeworks of the mature heavy chain variable region and SEQ ID NO:10is/are selected from the group consisting of H27 occupied by F, H28occupied by N, H48 occupied by I, H66 occupied by K, H67 occupied by A,H71 occupied by A, H76 occupied by N, H93 occupied by N, H94 occupied byV, L37 occupied by L, L39 occupied by K, L45 occupied by K, and L46occupied by L. Optionally, the 3 CDRs of the mature heavy chain variableregion are those of SEQ ID NO. 10 and the 3 CDRs of the mature lightchain variable region are those of SEQ ID NO:15. The CDRs are shown inFIG. 1. Optionally, the mature heavy chain variable region is fused to aheavy chain constant region and the mature light chain constant regionis fused to a light chain constant region. Optionally, the heavy chainconstant region is a mutant form of natural human constant region whichhas reduced binding to an Fcgamma receptor relative to the natural humanconstant region. Optionally, the heavy chain constant region is of IgG1isotype. Optionally, the heavy chain constant region has an amino acidsequence comprising SEQ ID NO:6 and the light chain constant region hasan amino acid sequence comprising SEQ ID NO:4. Optionally, the heavychain constant region has an amino acid sequence comprising SEQ ID NO:8(S239C) and the light chain constant region has an amino acid sequencecomprising SEQ ID NO:4. Optionally, any differences in CDRs of themature heavy chain variable region and mature light variable region fromSEQ ID NOS. 10 and 15 respectively reside in positions H60-H65.Optionally, the mature heavy chain variable region has an amino acidsequence comprising SEQ ID NO:10 and the mature light chain variableregion has an amino acid sequence comprising SEQ ID NO:15. Optionally,the antibody is conjugated to a cytotoxic or cytostatic agent. Preferredhumanized antibodies having greater affinity for LIV-1 than the antibodyBR2-14a. In another embodiment, the humanized antibody has anassociation constant for human or cynomolgus monkey LIV-1 of 0.5 to2×10⁹M⁻¹.

The invention also provides a humanized antibody comprising a matureheavy chain variable region comprising the three Kabat CDRs of SEQ IDNO:52, wherein position H27 is occupied by L, position H29 is occupiedby I, H30 by E, H76 by N, and H94 by V and a mature light chain variableregion comprising the three Kabat CDRs of SEQ ID NO:60 provided positionL36 is occupied by Y and position L46 by P. The invention also providesa nucleic acid encoding a mature heavy chain variable region and/or amature light chain variable region of any of the above defined humanizedantibodies.

The invention further provides a method of treating a patient having orat risk of cancer, comprising administering to the patient an effectiveregime of any of the above defined humanized antibodies. The cancer canbe for example a breast cancer, cervical cancer, melanoma, or a prostatecancer.

The invention further provides a pharmaceutical composition comprising ahumanized antibody as defined above.

The invention further provides methods of treating a subject afflictedwith a melanoma that expresses the LIV-1 protein by administering to thesubject a LIV-1 specific antibody or a LIV-1 antibody drug conjugate, inan amount sufficient to inhibit growth of the melanoma cancer cells.

The invention further provides methods of treating a subject afflictedwith a cervical cancer that expresses the LIV-1 protein by administeringto the subject a LIV-1 specific antibody or a LIV-1 antibody drugconjugate, in an amount sufficient to inhibit growth of the cervicalcancer cells.

The invention further provides a humanized antibody comprising a matureheavy chain variable region having an amino acid sequence at least 90%identical to HB (SEQ ID NO:10) and a mature light chain variable regionat least 90% identical to LB (SEQ ID NO:5). Optionally, the antibodycomprises a mature heavy chain variable region having an amino acidsequence at least 95% identical to HB and a mature light chain variableregion at least 95% identical to LB. Optionally, in any such antibody,positions H29, H30 and H76 are occupied by I, E and N, and L36 isoccupied by Y. Optionally, any difference in the variable regionframeworks of the mature heavy chain variable region and SEQ ID NO:10is/are selected from the group consisting of H27 occupied by F, H28occupied by N, H48 occupied by I, H66 occupied by K, H67 occupied by A,H71 occupied by A, H76 occupied by N, H93 occupied by N, H94 occupied byV, L37 occupied by L, L39 occupied by K, L45 occupied by K, and L46occupied by L. Optionally, the 3 CDRs of the mature heavy chain variableregion are those of SEQ ID NO. 10 and the 3 CDRs of the mature lightchain variable region are those of SEQ ID NO:15. The CDRs are shown inFIG. 1. Optionally, the mature heavy chain variable region is fused to aheavy chain constant region and the mature light chain constant regionis fused to a light chain constant region. Optionally, the heavy chainconstant region is a mutant form of natural human constant region whichhas reduced binding to an Fcgamma receptor relative to the natural humanconstant region. Optionally, the heavy chain constant region is of IgG1isotype. Optionally, the heavy chain constant region has an amino acidsequence comprising SEQ ID NO:6 and the light chain constant region hasan amino acid sequence comprising SEQ ID NO:4. Optionally, the heavychain constant region has an amino acid sequence comprising SEQ ID NO:8(S239C) and the light chain constant region has an amino acid sequencecomprising SEQ ID NO:4. Optionally, any differences in CDRs of themature heavy chain variable region and mature light variable region fromSEQ ID NOS. 10 and 15 respectively reside in positions H60-H65.Optionally, the mature heavy chain variable region has an amino acidsequence comprising SEQ ID NO:10 and the mature light chain variableregion has an amino acid sequence comprising SEQ ID NO:15. Optionally,the antibody is conjugated to a cytotoxic or cytostatic agent. Preferredhumanized antibodies having greater affinity for LIV-1 than the antibodyBR2-14a In another embodiment, the humanized antibody has an associationconstant for human or cynomolgus monkey LIV-1 of 0.5 to 2×10⁹ M⁻¹.

The invention further provides a humanized antibody comprising a matureheavy chain variable region comprising the 3 CDRs of SEQ ID NO:10 andwherein positions H29, H30 and H76 are occupied by I, E and Nrespectively, and a mature light chain variable region comprising the 3CDRs of SEQ ID NO:15, and wherein position L36 is occupied by Y.

The invention further provides a nucleic acid encoding a mature heavychain variable region and/or a mature light chain variable region of anyof the humanized antibodies described above.

The invention further provides a method of treating a patient having orat risk of cancer, comprising administering to the patient an effectiveregime of a humanized antibody as described above. Optionally, thecancer is breast cancer, cervical cancer, melanoma, or a prostatecancer.

The invention further provides a pharmaceutical composition comprising ahumanized antibody as described above.

The invention further provides a method of treating a patient having orat risk of triple negative breast cancer, comprising administering tothe patient an effective regime of an antibody that specifically bindsto LIV-1. Optionally, in such methods, the antibody is conjugated to acytotoxic or cytostatic agent.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an alignment of the amino acid sequences of humanized LIV-1heavy chain variable regions for hLIV-1 HA (SEQ ID NO:9), hLIV-1 HB (SEQID NO:10), hLIV-1 HC (SEQ ID NO:11), hLIV-1 HD (SEQ ID NO:12), andhLIV-1 HE (SEQ ID NO:13) with the heavy chain variable region of theparental murine mAb (referred to as BR2-14a) (SEQ ID NO:86) (upper twopanels). FIG. 1 further shows an alignment of the amino acid sequencesof humanized light chain variable regions for hLIV-1 LA (SEQ ID NO:14),hLIV-1 LB (SEQ ID NO:15), hLIV-1 LC (SEQ ID NO:16), hLIV-1 LD (SEQ IDNO:17), hLIV-1 LE (SEQ ID NO:18), and hLIV-1 LF (SEQ ID NO:19) with thelight chain variable region of the parental murine mAb (referred to asBR2-14a) (SEQ ID NO:87) (lower two panels).

FIG. 2 shows the binding curves for the humanized LIV-1 mAbs and theparental murine antibody (referred to as BR2-14a).

FIG. 3 shows the results of competition binding studies of the humanizedLIV-1 mAbs and the parental murine antibody (referred to as BR2-14a).The numbers in parentheses after each variant indicate the number ofback mutations.

FIG. 4 shows the results of saturation binding studies on MCF7 cells.BR2-14a-AF refers to AF-labeled parental murine antibody. hLIV-14 refersto AF-labeled HBLB antibody, a humanized antibody that specificallybinds to LIV-1.

FIG. 5 shows the results of competition binding studies on CHO cellsexpressing recombinant LIV-1 protein. BR2-14a refers to the parentalmurine antibody. hLIV-14 HBLB WT refers to the HBLB antibody. hLIV-14HBLB S239C refers to the HBLB antibody having serine to cysteinesubstitutions at each position in the heavy chain.

FIG. 6 shows an analysis of LIV-1 protein expression by IHC onpost-hormone treated breast cancer patient samples.

FIG. 7 shows an analysis of LIV-1 protein expression by IHC onhormone-refractory metastatic prostate cancer patient samples.

FIG. 8 shows an analysis of LIV-1 protein expression by IHC on triplenegative breast cancer patient samples.

FIG. 9 shows the results of cytotoxicity assays on hLIV-14 antibody drugconjugates, i.e., the HBLB mAb conjugated to vcMMAE (1006) or mcMMAF(1269), as well as conjugates of control murine (mIgG) and human (hIgG)antibodies. hLIV-14-SEA-1006 refers to a non-fucosylated form of theHBLB mAb conjugated to vcMMAE (1006).

FIG. 10 shows the results of an in vitro ADCC assay on MCF7 cells usinghuman NK cells (donor 1; V/V). hLIV-14 WT refers to the HBLB mAb.hLIV-14 SEA refers to the non-fucosylated form of the HBLB mAb. hLIV-14mcMMAF refers to an antibody drug conjugate of the HBLB mAb conjugatedto mcMMAF. hLIV-14 vcMMAE refers to an antibody drug conjugate of theHBLB mAb conjugated to vcMMAE. hLIV-14 SEA vcMMAE refers to anon-fucosylated form of the HBLB mAb-vcMMAE antibody drug conjugate.

FIG. 11 shows the results of an in vitro ADCC assay on MCF7 cells usinghuman NK cells (donor 2). hLIV-14 WT refers to the HBLB mAb. hLIV-14 SEArefers to the non-fucosylated form of the HBLB mAb. cLIV-14 SEA refersto the non-fucosylated form of the chimeric parental murine antibodyhLIV-14 mcF(4) refers to an antibody drug conjugate of the HBLB mAb withan average of 4 mcMMAF drug linker molecules per antibody. hLIV-14vcE(4) refers to an antibody drug conjugate of the HBLB mAb with anaverage of 4 vcMMAE drug linker molecules per antibody. hLIV-14 vcE(4)SEA refers to a non-fucosylated form of the HBLB mAb-vcMMAE antibodydrug conjugate having an average of four vcMMAE drug linker moleculesper antibody. hIgG refers to control human IgG. H00-mcF(4) refers to acontrol antibody drug conjugate of a nonbinding antibody with an averageof 4 mcMMAF drug linker molecules per antibody. H00-vcE(4) refers to acontrol antibody drug conjugate of a nonbinding antibody with an averageof 4 vcMMAE drug linker molecules per antibody.

FIG. 12 shows the results of a xenograft study of the MCF7 breast cancerline in nude mice. cLIV-14-mcMMAF(4) refers to an antibody drugconjugate of the chimeric form of the parental murine antibody having anaverage of 4 mcMMAF drug linker molecules per antibody.cLIV-14-vcMMAE(4) refers to an antibody drug conjugate of the chimericform of the parent murine antibody having an average of 4 vcMMAE druglinker molecules per antibody. H00-mcMMAF(4) refers to an antibody drugconjugate of a nonbinding control antibody having an average of 4 mcMMAFdrug linker molecules per antibody. H00-vcMMAE(4) refers to an antibodydrug conjugate of a nonbinding control antibody having an average of 4vcMMAE drug linker molecules per antibody. The dose and time ofadministration of indicated on the figure.

FIG. 13 shows the results of a xenograft study of the PC3 prostatecancer line in male nude mice. cLIV-14-vcMMAE(4) refers to an antibodydrug conjugate of the chimeric form of the parent murine antibody havingan average of 4 vcMMAE drug linker molecules per antibody.hBU12-vcMMAE(4) refers to an antibody drug conjugate of an anti-CD19antibody having an average of 4 vcMMAE drug linker molecules perantibody. The dose and time of administration of indicated on thefigure.

FIG. 14 shows the results of a xenograft study of the MCF7 breast cancerline in nude mice. hLIV-14-vcMMAE (4) refers to an antibody drugconjugate of the HBLB antibody having an average of 4 vcMMAE drug linkermolecules per antibody. hLIV-14d-vcMMAE (2) refers to an antibody drugconjugate of the HBLB antibody having an average of 2 vcMMAE drug linkermolecules per antibody, each conjugated at the S239C position of eachheavy chain. H00-vcMMAE(4) refers to an antibody drug conjugate of anonbinding control antibody having an average of 4 vcMMAE drug linkermolecules per antibody. The dose and time of administration of indicatedon the figure.

FIG. 15 shows the results of a xenograft study of the PC3 prostatecancer line in male nude mice. hLIV-14-vcMMAE (4) refers to an antibodydrug conjugate of the HBLB antibody having an average of 4 vcMMAE druglinker molecules per antibody. hLIV-14-mcMMAF(4) refers to an antibodydrug conjugate of the HBLB antibody having an average of 4 mcMMAF druglinker molecules per antibody. hLIV-14d-vcMMAE(2) refers to an antibodydrug conjugate of the HBLB antibody having an average of 2 vcMMAE druglinker molecules per antibody, each conjugated at the S239C position ofeach heavy chain. hLIV-14d-mcMMAF(2) refers to an antibody drugconjugate of the HBLB antibody having an average of 2 mcMMAF drug linkermolecules per antibody, each conjugated at the S239C position of eachheavy chain. H00-vcMMAE(4) refers to an antibody drug conjugate of anonbinding control antibody having an average of 4 vcMMAE drug linkermolecules per antibody. H00-mcMMAF(4) refers to an antibody drugconjugate of a nonbinding control antibody having an average of 4 mcMMAFdrug linker molecules per antibody. The dose and time of administrationof indicated on the figure.

FIGS. 16A and 16B show alignments of humanized heavy chain (FIG. 16A)and light chain (FIG. 16B) mature variable regions with those of themouse BR2-22a. FIG. 16A shows an alignment of the amino acid sequencesof humanized heavy chain variable regions for hLIV-22 HA (SEQ ID NO:47),hLIV-22 HB (SEQ ID NO:48), hLIV-22 HC (SEQ ID NO:49), hLIV-22 HD (SEQ IDNO:50), hLIV-22 HE (SEQ ID NO:51), hLIV-22 HF (SEQ ID NO:52), andhLIV-22 HG (SEQ ID NO:53) with the heavy chain variable region of theparental murine mAb (referred to as BR2-22a) (SEQ ID NO:88). FIG. 16Bshows an alignment of the amino acid sequences of humanized light chainvariable regions for hLIV-22 LA (SEQ ID NO:54), hLIV-22 LB (SEQ IDNO:55), hLIV-22 LC (SEQ ID NO:56), hLIV-22 LD (SEQ ID NO:57), hLIV-22 LE(SEQ ID NO:58), hLIV-22 LF (SEQ ID NO:59), and hLIV-22 LG (SEQ ID NO:60)with the light chain variable region of the parental murine mAb(referred to as BR2-22a) (SEQ ID NO:89).

FIG. 17 shows competition binding assays of different permutations ofhumanized heavy chains HA-HF and humanized light chains LA-LF derivedfrom the murine monoclonal anti LIV-1 antibody BR2-22a. The total numberof murine back mutations in each light or heavy chain is shown inparentheses. Only HELF showed sufficient retention of binding.

FIG. 18 shows systematic variation of the HE and LF chains to testcontribution of individual backmutations to antigen binding. Sites ofpotential somatic hypermutation are in parentheses. Mouse residues areunderlined. The remaining residues are human germline residues.

FIG. 19 shows competition binding of the LF variants on the top of thefigure. The tested back mutations are shown in the bottom of the figure.Mouse residues are underlined. The remaining residues are human germlineresidues.

FIG. 20 shows competition binding of the HE variants on the top of thefigure. The tested back mutations are shown in the bottom of the figure.Mouse residues are underlined. The remaining residues are human germlineresidues.

FIG. 21 shows competition binding of different permutations of HE, HF,HG and LF and LG.

FIG. 22 shows saturation binding of humanized LIV14 antibody andhumanized LIV22 antibody on human and cynomolgus LIV-1 expressed fromCHO cells.

FIG. 23 shows cytotoxic activity of humanized LIV22-vcMMAE on MCF-7cells after 144 hr of treatment. h00-1006 is a control drug-conjugatedantibody.

FIG. 24 shows cytotoxic activity of hLIV22-mcMMAF on MCF-7 cells after144 hr of treatment. h00-1269 is a control drug-conjugated antibody.

FIG. 25 shows the activity of hLIV22 antibody on PC3 (DSMZ) prostatecarcinoma model in nude female mice. Dose days are indicated bytriangles on the X-axis.

FIG. 26 shows that activity of hLIV22 antibody on MCF7 (NCI) breastcarcinoma tumors in nude mice.

FIG. 27 compares the activity of hLIV22 and hLIV14 in the same model asFIG. 26.

FIG. 28 shows an analysis of LIV-1 protein expression by IHC on melanomacancer patient samples.

DEFINITIONS

Monoclonal antibodies are typically provided in isolated form. Thismeans that an antibody is typically at least 50% w/w pure of interferingproteins and other contaminants arising from its production orpurification but does not exclude the possibility that the monoclonalantibody is combined with an excess of pharmaceutical acceptablecarrier(s) or other vehicle intended to facilitate its use. Sometimesmonoclonal antibodies are at least 60%, 70%, 80%, 90%, 95 or 99% w/wpure of interfering proteins and contaminants from production orpurification.

Specific binding of a monoclonal antibody to its target antigen means anaffinity of at least 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ M⁻¹. Specific bindingis detectably higher in magnitude and distinguishable from non-specificbinding occurring to at least one unrelated target. Specific binding canbe the result of formation of bonds between particular functional groupsor particular spatial fit (e.g., lock and key type) whereas nonspecificbinding is usually the result of van der Waals forces. Specific bindingdoes not however necessarily imply that a monoclonal antibody binds oneand only one target.

The basic antibody structural unit is a tetramer of subunits. Eachtetramer includes two identical pairs of polypeptide chains, each pairhaving one “light” (about 25 kDa) and one “heavy” chain (about 50-70kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. This variable region is initially expressed linkedto a cleavable signal peptide. The variable region without the signalpeptide is sometimes referred to as a mature variable region. Thus, forexample, a light chain mature variable region, means a light chainvariable region without the light chain signal peptide. Thecarboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function.

Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, and define theantibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 or more amino acids. (See generally,Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989,Ch. 7, incorporated by reference in its entirety for all purposes).

The mature variable regions of each light/heavy chain pair form theantibody binding site. Thus, an intact antibody has two binding sites.Except in bifunctional or bispecific antibodies, the two binding sitesare the same. The chains all exhibit the same general structure ofrelatively conserved framework regions (FR) joined by threehypervariable regions, also called complementarity determining regionsor CDRs. The CDRs from the two chains of each pair are aligned by theframework regions, enabling binding to a specific epitope. FromN-terminal to C-terminal, both light and heavy chains comprise thedomains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of aminoacids to each domain is in accordance with the definitions of Kabat,Sequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md., 1987 and 1991), or Chothia & Lesk, J. Mol. Biol.196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989). Kabatalso provides a widely used numbering convention (Kabat numbering) inwhich corresponding residues between different heavy chains or betweendifferent light chains are assigned the same number.

The term “antibody” includes intact antibodies and binding fragmentsthereof. Typically, antibody fragments compete with the intact antibodyfrom which they were derived for specific binding to the targetincluding separate heavy chains, light chains Fab, Fab′, F(ab′)₂,F(ab)c, diabodies, Dabs, nanobodies, and Fv. Fragments can be producedby recombinant DNA techniques, or by enzymatic or chemical separation ofintact immunoglobulins. The term “antibody” also includes a diabody(homodimeric Fv fragment) or a minibody (V_(L)-V_(H)-C_(H)3), abispecific antibody or the like. A bispecific or bifunctional antibodyis an artificial hybrid antibody having two different heavy/light chainpairs and two different binding sites (see, e.g., Songsivilai andLachmann, Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J.Immunol., 148:1547-53 (1992)). The term “antibody” includes an antibodyby itself (naked antibody) or an antibody conjugated to a cytotoxic orcytostatic drug.

The term “epitope” refers to a site on an antigen to which an antibodybinds. An epitope can be formed from contiguous amino acids ornoncontiguous amino acids juxtaposed by tertiary folding of one or moreproteins. Epitopes formed from contiguous amino acids are typicallyretained on exposure to denaturing solvents whereas epitopes formed bytertiary folding are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5 or 8-10 amino acids in a unique spatial conformation. Methods ofdetermining spatial conformation of epitopes include, for example, x-raycrystallography and 2-dimensional nuclear magnetic resonance. See, e.g.,Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66,Glenn E. Morris, Ed. (1996).

Antibodies that recognize the same or overlapping epitopes can beidentified in a simple immunoassay showing the ability of one antibodyto compete with the binding of another antibody to a target antigen. Theepitope of an antibody can also be defined by X-ray crystallography ofthe antibody bound to its antigen to identify contact residues.Alternatively, two antibodies have the same epitope if all amino acidmutations in the antigen that reduce or eliminate binding of oneantibody reduce or eliminate binding of the other. Two antibodies haveoverlapping epitopes if some amino acid mutations that reduce oreliminate binding of one antibody reduce or eliminate binding of theother.

Competition between antibodies is determined by an assay in which anantibody under test inhibits specific binding of a reference antibody toa common antigen (see, e.g., Junghans et al., Cancer Res. 50:1495,1990). A test antibody competes with a reference antibody if an excessof a test antibody (e.g., at least 2×, 5×, 10×, 20× or 100×) inhibitsbinding of the reference antibody by at least 50% but preferably 75%,90% or 99% as measured in a competitive binding assay. Antibodiesidentified by competition assay (competing antibodies) includeantibodies binding to the same epitope as the reference antibody andantibodies binding to an adjacent epitope sufficiently proximal to theepitope bound by the reference antibody for steric hindrance to occur.

The term “patient” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment.

For purposes of classifying amino acids substitutions as conservative ornonconservative, amino acids are grouped as follows: Group I(hydrophobic side chains): met, ala, val, leu, ile; Group II (neutralhydrophilic side chains): cys, ser, thr; Group III (acidic side chains):asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V(residues influencing chain orientation): gly, pro; and Group VI(aromatic side chains): trp, tyr, phe. Conservative substitutionsinvolve substitutions between amino acids in the same class.Non-conservative substitutions constitute exchanging a member of one ofthese classes for a member of another.

Percentage sequence identities are determined with antibody sequencesmaximally aligned by the Kabat numbering convention. After alignment, ifa subject antibody region (e.g., the entire mature variable region of aheavy or light chain) is being compared with the same region of areference antibody, the percentage sequence identity between the subjectand reference antibody regions is the number of positions occupied bythe same amino acid in both the subject and reference antibody regiondivided by the total number of aligned positions of the two regions,with gaps not counted, multiplied by 100 to convert to percentage.

Compositions or methods “comprising” one or more recited elements mayinclude other elements not specifically recited. For example, acomposition that comprises antibody may contain the antibody alone or incombination with other ingredients.

Designation of a range of values includes all integers within ordefining the range.

An antibody effector function refers to a function contributed by an Fcdomain(s) of an Ig. Such functions can be, for example,antibody-dependent cellular cytotoxicity, antibody-dependent cellularphagocytosis or complement-dependent cytotoxicity. Such function can beeffected by, for example, binding of an Fc effector domain(s) to an Fcreceptor on an immune cell with phagocytic or lytic activity or bybinding of an Fc effector domain(s) to components of the complementsystem. Typically, the effect(s) mediated by the Fc-binding cells orcomplement components result in inhibition and/or depletion of the LIV-1targeted cell. Fc regions of antibodies can recruit Fc receptor(FcR)-expressing cells and juxtapose them with antibody-coated targetcells. Cells expressing surface FcR for IgGs including FcγRIII (CD16),FcγRII (CD32) and FcγRIII (CD64) can act as effector cells for thedestruction of IgG-coated cells. Such effector cells include monocytes,macrophages, natural killer (NK) cells, neutrophils and eosinophils.Engagement of FcγR by IgG activates antibody-dependent cellularcytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP).ADCC is mediated by CD16⁺ effector cells through the secretion ofmembrane pore-forming proteins and proteases, while phagocytosis ismediated by CD32⁺ and CD64⁺ effector cells (see Fundamental Immunology,4^(th) ed., Paul ed., Lippincott-Raven, N.Y., 1997, Chapters 3, 17 and30; Uchida et al., 2004, J. Exp. Med. 199:1659-69; Akewanlop et al.,2001, Cancer Res. 61:4061-65; Watanabe et al., 1999, Breast Cancer Res.Treat. 53:199-207). In addition to ADCC and ADCP, Fc regions ofcell-bound antibodies can also activate the complement classical pathwayto elicit complement-dependent cytotoxicity (CDC). C1q of the complementsystem binds to the Fc regions of antibodies when they are complexedwith antigens. Binding of C1q to cell-bound antibodies can initiate acascade of events involving the proteolytic activation of C4 and C2 togenerate the C3 convertase. Cleavage of C3 to C3b by C3 convertaseenables the activation of terminal complement components including C5b,C6, C7, C8 and C9. Collectively, these proteins form membrane-attackcomplex pores on the antibody-coated cells. These pores disrupt the cellmembrane integrity, killing the target cell (see Immunobiology, 6^(th)ed., Janeway et al., Garland Science, N. Y., 2005, Chapter 2).

The term “antibody-dependent cellular cytotoxicity”, or ADCC, is amechanism for inducing cell death that depends upon the interaction ofantibody-coated target cells with immune cells possessing lytic activity(also referred to as effector cells). Such effector cells includenatural killer cells, monocytes/macrophages and neutrophils. Theeffector cells attach to an Fc effector domain(s) of Ig bound to targetcells via their antigen-combining sites. Death of the antibody-coatedtarget cell occurs as a result of effector cell activity.

The term “antibody-dependent cellular phagocytosis”, or ADCP, refers tothe process by which antibody-coated cells are internalized, either inwhole or in part, by phagocytic immune cells (e.g., macrophages,neutrophils and dendritic cells) that bind to an Fc effector domain(s)of Ig.

The term “complement-dependent cytotoxicity”, or CDC, refers to amechanism for inducing cell death in which an Fc effector domain(s) of atarget-bound antibody activates a series of enzymatic reactionsculminating in the formation of holes in the target cell membrane.Typically, antigen-antibody complexes such as those on antibody-coatedtarget cells bind and activate complement component C1q which in turnactivates the complement cascade leading to target cell death.Activation of complement may also result in deposition of complementcomponents on the target cell surface that facilitate ADCC by bindingcomplement receptors (e.g., CR3) on leukocytes.

A “cytotoxic effect” refers to the depletion, elimination and/or thekilling of a target cell. A “cytotoxic agent” refers to an agent thathas a cytotoxic effect on a cell. Cytotoxic agents can be conjugated toan antibody or administered in combination with an antibody.

A “cytostatic effect” refers to the inhibition of cell proliferation. A“cytostatic agent” refers to an agent that has a cytostatic effect on acell, thereby inhibiting the growth and/or expansion of a specificsubset of cells. Cytostatic agents can be conjugated to an antibody oradministered in combination with an antibody.

The term “pharmaceutically acceptable” means approved or approvable by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “pharmaceuticallycompatible ingredient” refers to a pharmaceutically acceptable diluent,adjuvant, excipient, or vehicle with which an anti-LIV-1 antibody.

The phrase “pharmaceutically acceptable salt,” refers topharmaceutically acceptable organic or inorganic salts of an anti-LIV-1antibody or conjugate thereof or agent administered with an anti-LIV-1antibody. Exemplary salts include sulfate, citrate, acetate, oxalate,chloride, bromide, iodide, nitrate, bisulfate, phosphate, acidphosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate,oleate, tannate, pantothenate, bitartrate, ascorbate, succinate,maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate,formate, benzoate, glutamate, methanesulfonate, ethanesulfonate,benzenesulfonate, p toluenesulfonate, and pamoate (i.e., 1,1′ methylenebis-(2 hydroxy 3 naphthoate)) salts. A pharmaceutically acceptable saltmay involve the inclusion of another molecule such as an acetate ion, asuccinate ion or other counterion. The counterion may be any organic orinorganic moiety that stabilizes the charge on the parent compound.Furthermore, a pharmaceutically acceptable salt may have more than onecharged atom in its structure. Instances where multiple charged atomsare part of the pharmaceutically acceptable salt can have multiplecounter ions. Hence, a pharmaceutically acceptable salt can have one ormore charged atoms and/or one or more counterion.

Unless otherwise apparent from the context, the term “about” encompassesvalues within a standard deviation of a stated value.

DETAILED DESCRIPTION

I. General

The invention provides monoclonal antibodies that specifically bind toLIV-1. The antibodies are useful for treatment and diagnoses of variouscancers as well as detecting LIV-1.

II. Target Molecules

Unless otherwise indicated, LIV-1 means a human LIV-1. An exemplaryhuman sequence is assigned Swiss Prot accession number Q13433. Q13433 isincluded herein as SEQ ID NO:83. Three variant isoforms and onepolymorphism are known. A second version of the human LIV-1 protein,accession number AAA96258.2, is included herein as SEQ ID NO:84. Fourextracellular domains are bounded by residues 29-325, 377-423, 679-686and 746-755 of Q13433 respectively.

Unless otherwise apparent from the context reference LIV-1 means atleast an extracellular domain of the protein and usually the completeprotein other than a cleavable signal peptide (amino acids 1-28 ofQ13433).

III. Antibodies of the Invention

A. Binding Specificity and Functional Properties

The invention provides humanized antibodies derived from two mouseantibodies, BR2-14a and BR2-22a. Unless specifically indicatedotherwise, the present disclosures relate to both antibodies. The twomouse antibodies show 94% and 91% sequence identity to one another inthe mature heavy and light chain variable regions. The two antibodiesbind to the same or overlapping epitopes on human LIV-1. However, theBR2-22a antibody has about ten-fold higher affinity for human LIV-1 andabout 3-fold higher affinity for cynomolgus monkey LIV-1 than BR2-14a asshown in FIG. 22.

The affinity of humanized forms of the mouse BR2-14a antibody (i.e., Ka)is preferably within a factor of five or a factor of two of that of themouse antibody BR2-14a for human LIV-1. Humanized BR2-14a antibodiesspecifically bind to human LIV-1 in native form and/or recombinantlyexpressed from CHO cells as does the mouse antibody from which they werederived. Preferred humanized BR2-14a antibodies have an affinity thesame as or greater than (i.e., greater than beyond margin of error inmeasurement) that of BR2-14a for human LIV-1 (e.g., 1.1-5 fold, 1.1 to 3fold, 1.5 to 3-fold, 1.7 to 2.3-fold or 1.7-2.1-fold the affinity orabout twice the affinity of BR2-14a). Preferred humanized BR2-14aantibodies bind to the same epitope and/or compete with BR2-14a forbinding to human LIV-1. Preferred humanized BR2-14a antibodies also bindto the cyno-homolog of LIV-1 thus permitting preclinical testing innonhuman primates.

The affinity of humanized forms of the mouse BR2-22a antibody (i.e., Ka)for human LIV-1, natively expressed or expressed from CHO cells, ispreferably within a factor of five or a factor of two of that of themouse antibody BR2-22. Some humanized BR2-22a antibodies have anassociation constant that is essentially the same as that of BR2-22a(i.e., within experimental error). Some humanized BR2-22a antibodieshave an association constant within a range of 0.5 to 1 or 0.5-1.5 thatof the association constant of the BR2-22a antibody. Preferred humanizedBR2-22a antibodies have an association constant greater than 5×10⁸ M⁻¹,or in a range of 0.5 to 2×10⁹ M⁻¹ or about 0.8×10⁹ M⁻¹ (+/− error inmeasurement) for human LIV-1 expressed from CHO cells. Here as elsewherein this application, affinities can be measured in accordance with themethods of the Examples. Preferred humanized BR2-22a antibodies bind tothe same epitope and/or compete with BR2-22a for binding to human LIV-1.Humanized BR2-22a antibodies bind to the cyno-homolog of LIV-1 as wellas human LIV-1. Preferred humanized BR2-22a antibodies bind withessentially the same association constant to human and cynomolgus monkeyLIV-1 both expressed from CHO cells (within experimental error) thuspermitting and increasing the predictive accuracy of preclinical testingin nonhuman primates.

Preferred antibodies (both humanized BR2-14a and humanized BR2-22a)inhibit cancer (e.g., growth of cells, metastasis and/or lethality tothe organisms) as shown on cancerous cells propagating in culture, in ananimal model or clinical trial. Animal models can be formed byimplanting LIV-1-expressing human tumor cell lines into appropriateimmunodeficient rodent strains, e.g., athymic nude mice or SCID mice.These tumor cell lines can be established in immunodeficient rodenthosts either as solid tumor by subcutaneous injections or asdisseminated tumors by intravenous injections. Once established within ahost, these tumor models can be applied to evaluate the therapeuticefficacies of the anti-LIV-1 antibodies or conjugated forms thereof asdescribed in the Examples.

B. Humanized Antibodies

A humanized antibody is a genetically engineered antibody in which theCDRs from a non-human “donor” antibody are grafted into human “acceptor”antibody sequences (see, e.g., Queen, U.S. Pat. Nos. 5,530,101 and5,585,089; Winter, U.S. Pat. No. 5,225,539; Carter, U.S. Pat. No.6,407,213; Adair, U.S. Pat. No. 5,859,205; and Foote, U.S. Pat. No.6,881,557). The acceptor antibody sequences can be, for example, amature human antibody sequence, a composite of such sequences, aconsensus sequence of human antibody sequences, or a germline regionsequence. A preferred acceptor sequence for the heavy chain is thegermline V_(H) exon V_(H)1-2 (also referred to in the literature asHV1-2) (Shin et al., 1991, EMBO J. 10:3641-3645) and for the hingeregion (J_(H)), exon J_(H)-6 (Mattila et al., 1995, Eur. J. Immunol.25:2578-2582). For the light chain, a preferred acceptor sequence isexon VK2-30 (also referred to in the literature as KV2-30) and for thehinge region exon Jκ-4 (Hieter et al., 1982, J. Biol. Chem.257:1516-1522). Thus, a humanized antibody is an antibody having some orall CDRs entirely or substantially from a donor antibody and variableregion framework sequences and constant regions, if present, entirely orsubstantially from human antibody sequences. Similarly a humanized heavychain has at least one, two and usually all three CDRs entirely orsubstantially from a donor antibody heavy chain, and a heavy chainvariable region framework sequence and heavy chain constant region, ifpresent, substantially from human heavy chain variable region frameworkand constant region sequences. Similarly a humanized light chain has atleast one, two and usually all three CDRs entirely or substantially froma donor antibody light chain, and a light chain variable regionframework sequence and light chain constant region, if present,substantially from human light chain variable region framework andconstant region sequences. Other than nanobodies and dAbs, a humanizedantibody comprises a humanized heavy chain and a humanized light chain.A CDR in a humanized antibody is substantially from a corresponding CDRin a non-human antibody when at least 60%, 85%, 90%, 95% or 100% ofcorresponding residues (as defined by Kabat) are identical between therespective CDRs. The variable region framework sequences of an antibodychain or the constant region of an antibody chain are substantially froma human variable region framework sequence or human constant regionrespectively when at least 85%, 90%, 95% or 100% of correspondingresidues defined by Kabat are identical.

Although humanized antibodies often incorporate all six CDRs (preferablyas defined by Kabat) from a mouse antibody, they can also be made withless than all CDRs (e.g., at least 3, 4, or 5) CDRs from a mouseantibody (e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos etal., Journal of Molecular Biology, 320: 415-428, 2002; Iwahashi et al.,Mol. Immunol. 36:1079-1091, 1999; Tamura et al, Journal of Immunology,164:1432-1441, 2000).

Certain amino acids from the human variable region framework residuescan be selected for substitution based on their possible influence onCDR conformation and/or binding to antigen. Investigation of suchpossible influences is by modeling, examination of the characteristicsof the amino acids at particular locations, or empirical observation ofthe effects of substitution or mutagenesis of particular amino acids.

For example, when an amino acid differs between a murine variable regionframework residue and a selected human variable region frameworkresidue, the human framework amino acid can be substituted by theequivalent framework amino acid from the mouse antibody when it isreasonably expected that the amino acid:

-   -   (1) noncovalently binds antigen directly,    -   (2) is adjacent to a CDR region,    -   (3) otherwise interacts with a CDR region (e.g. is within about        6 Å of a CDR region); or    -   (4) mediates interaction between the heavy and light chains.

The invention provides humanized forms of the mouse BR2-14a antibodyincluding five exemplified humanized heavy chain mature variable regions(HA-HE) and six exemplified humanized light chain mature variableregions (LA-LF). The permutations of these chains having the strongestbinding (lowest EC50) are HBLB, HBLF, HCLB, HCLF, HDLB, HDLF, HELE andHELF. Of these permutations, HBLB (also known as hLIV14) is preferredbecause it has the strongest binding, about 2 fold stronger than themouse donor antibody, and has the fewest back mutations (four).

The invention provides variants of the HBLB humanized antibody in whichthe humanized heavy chain mature variable region shows at least 90%, 95%or 99% identity to SEQ ID NO:10 and the humanized light chain maturevariable region shows at least 90%, 95% or 99% sequence identity to SEQID NO:15. Preferably, in such antibodies some or all of thebackmutations in HBLB are retained. In other words, at least 1, 2 orpreferably all 3 of heavy chain positions H29, H30 and H76 are occupiedby I and E and N, respectively. Likewise position L36 is preferablyoccupied by Y. The CDR regions of such humanized antibodies arepreferably substantially identical to the CDR regions of HBLB, which arethe same as those of the mouse donor antibody. The CDR regions can bedefined by any conventional definition (e.g., Chothia) but arepreferably as defined by Kabat. In one embodiment, the humanizedantibody comprises a heavy chain comprising the 3 CDRs of SEQ ID NO:10and variable region frameworks with at least 95% identity to thevariable region frameworks of SEQ ID NO:10. In another embodiment, thehumanized antibody comprises a light chain comprising the 3 CDRs of SEQID NO:15 and variable region frameworks with at least 95% identity tovariable region frameworks of SEQ ID NO:15. In a further embodiment, thehumanized antibody comprises a heavy chain comprising the 3 CDRs of SEQID NO:10 and variable region frameworks with at least 95% identity tothe variable region frameworks of SEQ ID NO:10, and a light chaincomprising the 3 CDRs of SEQ ID NO:15, and variable region frameworkswith at least 95% identity to the variable region frameworks of SEQ IDNO:15.

Insofar as humanized antibodies show any variation from the exemplifiedHBLB humanized antibody, one possibility for such additional variationis additional backmutations in the variable region frameworks. Any orall of the positions backmutated in other exemplified humanized heavy orlight chain mature variable regions can also be made (i.e., 1, 2, 3, 4,5, 6, 7, 8 or all 9 of H27 occupied by F, H28 occupied by N, H48occupied by I, H66 occupied by K, H67 occupied by A, H71 occupied by A,H76 occupied by N, H93 occupied by N and H94 occupied by V in the heavychain and 1, 2, 3, 4 or all 5 of L37 occupied by L, L39 occupied by K,L45 occupied by K, and L46 occupied by L in the light chain. However,such additional backmutations are not preferred because they in generaldo not improve affinity and introducing more mouse residues may giveincreased risk of immunogenicity.

The invention provides humanized forms of the mouse BR2-22a antibodyincluding three exemplified humanized heavy chain mature variableregions (HE, HF and HG) and two exemplified humanized light chain (LFand LG) which can be combined in different permutations with adequatebinding (see FIG. 21). Of these permutations, HGLG (also known ashLIV22) is preferred because it has the best combination of bindingproperties (essentially the same as the mouse BR2-22a antibody withinexperimental error) and fewest back mutations (seven).

The invention provides variants of the HGLG humanized antibody in whichthe humanized heavy chain mature variable region shows at least 90%,95%, 98% or 99% identity to SEQ ID NO:53 and the humanized light chainmature variable region shows at least 90%, 95%, 98% or 99% sequenceidentity to SEQ ID NO:60. Preferably, in such antibodies some or all ofthe backmutations in HGLG are retained. In other words, at least 1, 2,3, 4 or preferably all 5 of heavy chain positions H27, H29, H30, H76,and H94 are occupied by L, I, E, N and V (here, as elsewhere in thisapplication Kabat numbering is used to describe positions in the maturevariable heavy and light chain variable regions). Of thesebackmutations, H94 contributes the most to retention of binding affinityand H76 the least. Likewise positions L36 and L46 are preferablyoccupied by Y and P respectively. The CDR regions of such humanizedantibodies are preferably substantially identical to the CDR regions ofHGLG, which are the same as those of the mouse donor antibody. The CDRregions can be defined by any conventional definition (e.g., Chothia)but are preferably as defined by Kabat. In one embodiment, the humanizedantibody comprises a heavy chain comprising the 3 CDRs of SEQ ID NO:53and variable region frameworks with at least 95% identity to thevariable region frameworks of SEQ ID NO:53. In another embodiment, thehumanized antibody comprises a light chain comprising the 3 CDR's of SEQID NO:60 and variable region frameworks with at least 95% identity tothe variable region frameworks of SEQ ID NO:60. In a further embodiment,the humanized antibody comprises a heavy chain comprising the 3 CDRs ofSEQ ID NO:53 and variable region frameworks with at least 95% identityto the variable region frameworks of SEQ ID NO:53, and a light chaincomprising the 3 CDRs of SEQ ID NO:60, and variable region frameworkswith at least 95% identity to the variable region frameworks of SEQ IDNO:60.

Insofar as humanized BR2-22a antibodies show any variation from theexemplified HGLG humanized antibody, one possibility for such additionalvariation is additional backmutations in the variable region frameworks.Any or all of the positions backmutated in other exemplified humanizedheavy or light chain mature variable regions can also be made (i.e., 1,2, 3, 4, 5, or all 6, of H28 occupied by N, H48 occupied by I, H66occupied by K, H67 occupied by A, H71 occupied by A, H93 occupied by Tin the heavy chain and 1 or, 2 of L37 occupied by L37 occupied by L, andL45 occupied by K. However, such additional backmutations are notpreferred because they in general do not improve affinity andintroducing more mouse residues may give increased risk ofimmunogenicity.

Another possible variation is to substitute certain residues in the CDRsof the mouse antibody with corresponding residues from human CDRssequences, typically from the CDRs of the human acceptor sequences usedin designing the exemplified humanized antibodies. In some antibodiesonly part of the CDRs, namely the subset of CDR residues required forbinding, termed the SDRs, are needed to retain binding in a humanizedantibody. CDR residues not contacting antigen and not in the SDRs can beidentified based on previous studies (for example residues H60-H65 inCDR H2 are often not required), from regions of Kabat CDRs lying outsideChothia hypervariable loops (Chothia, J. Mol. Biol. 196:901, 1987), bymolecular modeling and/or empirically, or as described in Gonzales etal., Mol. Immunol. 41: 863 (2004). In such humanized antibodies atpositions in which one or more donor CDR residues is absent or in whichan entire donor CDR is omitted, the amino acid occupying the positioncan be an amino acid occupying the corresponding position (by Kabatnumbering) in the acceptor antibody sequence. The number of suchsubstitutions of acceptor for donor amino acids in the CDRs to includereflects a balance of competing considerations. Such substitutions arepotentially advantageous in decreasing the number of mouse amino acidsin a humanized antibody and consequently decreasing potentialimmunogenicity. However, substitutions can also cause changes ofaffinity, and significant reductions in affinity are preferably avoided.In a further variation, one or more residues in a CDR of a humanizedBR2-22a antibody (which would otherwise be the same as the CDR of themouse BR2-22a antibody) can be replaced by corresponding residues from aCDR from the mouse BR2-14a antibody (or vice versa). Positions forsubstitution within CDRs and amino acids to substitute can also beselected empirically.

Although not preferred other amino acid substitutions can be made, forexample, in framework residues not in contact with the CDRs, or evensome potential CDR-contact residues amino acids within the CDRs. Oftenthe replacements made in the variant humanized sequences areconservative with respect to the replaced HBLB amino acids (in the caseof humanized BR2-14a) or HGLG amino acids (in the case of humanizedBR2-22). Preferably, replacements relative to HBLB or HGLG (whether ornot conservative) have no substantial effect on the binding affinity orpotency of the humanized mAb, that is, its ability to bind human LIV-1and inhibit growth of cancer cells.

Variants typically differ from the heavy and light chain mature variableregion sequences of HBLB (hLIV14) or HGLG (hLIV22) by a small number(e.g., typically no more than 1, 2, 3, 5 or 10 in either the light chainor heavy chain mature variable region, or both) of replacements,deletions or insertions.

C. Selection of Constant Region

The heavy and light chain variable regions of humanized antibodies canbe linked to at least a portion of a human constant region. The choiceof constant region depends, in part, whether antibody-dependentcell-mediated cytotoxicity, antibody dependent cellular phagocytosisand/or complement dependent cytotoxicity are desired. For example, humanisotopes IgG1 and IgG3 have strong complement-dependent cytotoxicity,human isotype IgG2 weak complement-dependent cytotoxicity and human IgG4lacks complement-dependent cytotoxicity. Human IgG1 and IgG3 also inducestronger cell mediated effector functions than human IgG2 and IgG4.Light chain constant regions can be lambda or kappa. Antibodies can beexpressed as tetramers containing two light and two heavy chains, asseparate heavy chains, light chains, as Fab, Fab′, F(ab′)2, and Fv, oras single chain antibodies in which heavy and light chain variabledomains are linked through a spacer.

Human constant regions show allotypic variation and isoallotypicvariation between different individuals, that is, the constant regionscan differ in different individuals at one or more polymorphicpositions. Isoallotypes differ from allotypes in that sera recognizingan isoallotype binds to a non-polymorphic region of a one or more otherisotypes.

One or several amino acids at the amino or carboxy terminus of the lightand/or heavy chain, such as the C-terminal lysine of the heavy chain,may be missing or derivatized in a proportion or all of the molecules.Substitutions can be made in the constant regions to reduce or increaseeffector function such as complement-mediated cytotoxicity or ADCC (see,e.g., Winter et al., U.S. Pat. No. 5,624,821; Tso et al., U.S. Pat. No.5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006),or to prolong half-life in humans (see, e.g., Hinton et al., J. Biol.Chem. 279:6213, 2004).

Exemplary substitution include the amino acid substitution of the nativeamino acid to a cysteine residue is introduced at amino acid position234, 235, 237, 239, 267, 298, 299, 326, 330, or 332, preferably an S239Cmutation in a human IgG1 isotype (US 20100158909). The presence of anadditional cysteine residue allows interchain disulfide bond formation.Such interchain disulfide bond formation can cause steric hindrance,thereby reducing the affinity of the Fc region-FcγR binding interaction.The cysteine residue(s) introduced in or in proximity to the Fc regionof an IgG constant region can also serve as sites for conjugation totherapeutic agents (i.e., coupling cytotoxic drugs using thiol specificreagents such as maleimide derivatives of drugs. The presence of atherapeutic agent causes steric hindrance, thereby further reducing theaffinity of the Fc region-FcγR binding interaction. Other substitutionsat any of positions 234, 235, 236 and/or 237 reduce affinity for Fcγreceptors, particularly FcγRI receptor (see, e.g., U.S. Pat. No.6,624,821, U.S. Pat. No. 5,624,821.)

The in vivo half-life of an antibody can also impact on its effectorfunctions. The half-life of an antibody can be increased or decreased tomodify its therapeutic activities. FcRn is a receptor that isstructurally similar to MHC Class I antigen that noncovalentlyassociates with β2-microglobulin. FcRn regulates the catabolism of IgGsand their transcytosis across tissues (Ghetie and Ward, 2000, Annu. Rev.Immunol. 18:739-766; Ghetie and Ward, 2002, Immunol. Res. 25:97-113).The IgG-FcRn interaction takes place at pH 6.0 (pH of intracellularvesicles) but not at pH 7.4 (pH of blood); this interaction enables IgGsto be recycled back to the circulation (Ghetie and Ward, 2000, Ann. Rev.Immunol. 18:739-766; Ghetie and Ward, 2002, Immunol. Res. 25:97-113).The region on human IgG1 involved in FcRn binding has been mapped(Shields et al., 2001, J. Biol. Chem. 276:6591-604). Alaninesubstitutions at positions Pro238, Thr256, Thr307, Gln311, Asp312,Glu380, Glu382, or Asn434 of human IgG1 enhance FcRn binding (Shields etal., 2001, J. Biol. Chem. 276:6591-604). IgG1 molecules harboring thesesubstitutions have longer serum half-lives. Consequently, these modifiedIgG1 molecules may be able to carry out their effector functions, andhence exert their therapeutic efficacies, over a longer period of timecompared to unmodified IgG1. Other exemplary substitutions forincreasing binding to FcRn include a Gln at position 250 and/or a Leu atposition 428. EU numbering is used for all position in the constantregion.

Oligosaccharides covalently attached to the conserved Asn297 areinvolved in the ability of the Fc region of an IgG to bind FcγR (Lund etal., 1996, J. Immunol. 157:4963-69; Wright and Morrison, 1997, TrendsBiotechnol. 15:26-31). Engineering of this glycoform on IgG cansignificantly improve IgG-mediated ADCC. Addition of bisectingN-acetylglucosamine modifications (Umana et al., 1999, Nat. Biotechnol.17:176-180; Davies et al., 2001, Biotech. Bioeng. 74:288-94) to thisglycoform or removal of fucose (Shields et al., 2002, J. Biol. Chem.277:26733-40; Shinkawa et al., 2003, J. Biol. Chem. 278:6591-604; Niwaet al., 2004, Cancer Res. 64:2127-33) from this glycoform are twoexamples of IgG Fc engineering that improves the binding between IgG Fcand FcγR, thereby enhancing Ig-mediated ADCC activity.

A systemic substitution of solvent-exposed amino acids of human IgG1 Fcregion has generated IgG variants with altered FcγR binding affinities(Shields et al., 2001, J. Biol. Chem. 276:6591-604). When compared toparental IgG1, a subset of these variants involving substitutions atThr256/Ser298, Ser298/Glu333, Ser298/Lys334, or Ser298/Glu333/Lys334 toAla demonstrate increased in both binding affinity toward FcγR and ADCCactivity (Shields et al., 2001, J. Biol. Chem. 276:6591-604; Okazaki etal., 2004, J. Mol. Biol. 336:1239-49).

Complement fixation activity of antibodies (both C1q binding and CDCactivity) can be improved by substitutions at Lys326 and Glu333(Idusogie et al., 2001, J. Immunol. 166:2571-2575). The samesubstitutions on a human IgG2 backbone can convert an antibody isotypethat binds poorly to C1q and is severely deficient in complementactivation activity to one that can both bind C1q and mediate CDC(Idusogie et al., 2001, J. Immunol. 166:2571-75). Several other methodshave also been applied to improve complement fixation activity ofantibodies. For example, the grafting of an 18-amino acidcarboxyl-terminal tail piece of IgM to the carboxyl-termini of IgGgreatly enhances their CDC activity. This is observed even with IgG4,which normally has no detectable CDC activity (Smith et al., 1995, J.Immunol. 154:2226-36). Also, substituting Ser444 located close to thecarboxy-terminal of IgG1 heavy chain with Cys induced tail-to-taildimerization of IgG1 with a 200-fold increase of CDC activity overmonomeric IgG1 (Shopes et al., 1992, J. Immunol. 148:2918-22). Inaddition, a bispecific diabody construct with specificity for C1q alsoconfers CDC activity (Kontermann et al., 1997, Nat. Biotech. 15:629-31).

Complement activity can be reduced by mutating at least one of the aminoacid residues 318, 320, and 322 of the heavy chain to a residue having adifferent side chain, such as Ala. Other alkyl-substituted non-ionicresidues, such as Gly, Ile, Leu, or Val, or such aromatic non-polarresidues as Phe, Tyr, Trp and Pro in place of any one of the threeresidues also reduce or abolish C1q binding. Ser, Thr, Cys, and Met canbe used at residues 320 and 322, but not 318, to reduce or abolish C1qbinding activity. Replacement of the 318 (Glu) residue by a polarresidue may modify but not abolish C1q binding activity. Replacingresidue 297 (Asn) with Ala results in removal of lytic activity but onlyslightly reduces (about three fold weaker) affinity for C1q. Thisalteration destroys the glycosylation site and the presence ofcarbohydrate that is required for complement activation. Any othersubstitution at this site also destroys the glycosylation site. Thefollowing mutations and any combination thereof also reduce C1q binding:D270A, K322A, P329A, and P311S (see WO 06/036291).

Reference to a human constant region includes a constant region with anynatural allotype or any permutation of residues occupying polymorphicpositions in natural allotypes. Also, up to 1, 2, 5, or 10 mutations maybe present relative to a natural human constant region, such as thoseindicated above to reduce Fcgamma receptor binding or increase bindingto FcRN.

D. Expression of Recombinant Antibodies

Humanized antibodies are typically produced by recombinant expression.Recombinant polynucleotide constructs typically include an expressioncontrol sequence operably linked to the coding sequences of antibodychains, including naturally-associated or heterologous promoter regions.Preferably, the expression control sequences are eukaryotic promotersystems in vectors capable of transforming or transfecting eukaryotichost cells. Once the vector has been incorporated into the appropriatehost, the host is maintained under conditions suitable for high levelexpression of the nucleotide sequences, and the collection andpurification of the crossreacting antibodies.

Mammalian cells are a preferred host for expressing nucleotide segmentsencoding immunoglobulins or fragments thereof. See Winnacker, From Genesto Clones, (VCH Publishers, N Y, 1987). A number of suitable host celllines capable of secreting intact heterologous proteins have beendeveloped in the art, and include CHO cell lines (e.g., DG44), variousCOS cell lines, HeLa cells, HEK293 cells, L cells, andnon-antibody-producing myelomas including Sp2/0 and NS0. Preferably, thecells are nonhuman. Expression vectors for these cells can includeexpression control sequences, such as an origin of replication, apromoter, an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), andnecessary processing information sites, such as ribosome binding sites,RNA splice sites, polyadenylation sites, and transcriptional terminatorsequences. Preferred expression control sequences are promoters derivedfrom endogenous genes, cytomegalovirus, SV40, adenovirus, bovinepapillomavirus, and the like. See Co et al., J. Immunol. 148:1149(1992).

Once expressed, antibodies can be purified according to standardprocedures of the art, including HPLC purification, columnchromatography, gel electrophoresis and the like (see generally, Scopes,Protein Purification (Springer-Verlag, N Y, 1982)).

IV. Nucleic Acids

The invention further provides nucleic acids encoding any of thehumanized heavy and light chains described above. Typically, the nucleicacids also encode a signal peptide fused to the mature heavy and lightchains. Coding sequences on nucleic acids can be in operable linkagewith regulatory sequences to ensure expression of the coding sequences,such as a promoter, enhancer, ribosome binding site, transcriptiontermination signal and the like. The nucleic acids encoding heavy andlight chains can occur in isolated form or can be cloned into one ormore vectors. The nucleic acids can be synthesized by for example, solidstate synthesis or PCR of overlapping oligonucleotides. Nucleic acidsencoding heavy and light chains can be joined as one contiguous nucleicacid, e.g., within an expression vector, or can be separate, e.g., eachcloned into its own expression vector.

V. Antibody Drug Conjugates

Anti-LIV-1 antibodies can be conjugated to cytotoxic or cytostaticmoieties (including pharmaceutically compatible salts thereof) to forman antibody drug conjugate (ADC). Particularly suitable moieties forconjugation to antibodies are cytotoxic agents (e.g., chemotherapeuticagents), prodrug converting enzymes, radioactive isotopes or compounds,or toxins (these moieties being collectively referred to as atherapeutic agent). For example, an anti-LIV-1 antibody can beconjugated to a cytotoxic agent such as a chemotherapeutic agent, or atoxin (e.g., a cytostatic or cytocidal agent such as, e.g., abrin, ricinA, pseudomonas exotoxin, or diphtheria toxin).

An anti-LIV-1 antibody can be conjugated to a pro-drug convertingenzyme. The pro-drug converting enzyme can be recombinantly fused to theantibody or chemically conjugated thereto using known methods. Exemplarypro-drug converting enzymes are carboxypeptidase G2, beta-glucuronidase,penicillin-V-amidase, penicillin-G-amidase, β-lactamase, β-glucosidase,nitroreductase and carboxypeptidase A.

Techniques for conjugating therapeutic agents to proteins, and inparticular to antibodies, are well-known. (See, e.g., Arnon et al.,“Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,”in Monoclonal Antibodies And Cancer Therapy (Reisfeld et al. eds., AlanR. Liss, Inc., 1985); Hellstrom et al., “Antibodies For Drug Delivery,”in Controlled Drug Delivery (Robinson et al. eds., Marcel Dekker, Inc.,2nd ed. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In CancerTherapy: A Review,” in Monoclonal Antibodies '84: Biological AndClinical Applications (Pinchera et al. eds., 1985); “Analysis, Results,and Future Prospective of the Therapeutic Use of Radiolabeled AntibodyIn Cancer Therapy,” in Monoclonal Antibodies For Cancer Detection AndTherapy (Baldwin et al. eds., Academic Press, 1985); and Thorpe et al.,1982, Immunol. Rev. 62:119-58. See also, e.g., PCT publication WO89/12624.)

The therapeutic agent can be conjugated in a manner that reduces itsactivity unless it is cleaved off the antibody (e.g., by hydrolysis, byantibody degradation or by a cleaving agent). Such therapeutic agent isattached to the antibody with a cleavable linker that is sensitive tocleavage in the intracellular environment of the LIV-1-expressing cancercell but is not substantially sensitive to the extracellularenvironment, such that the conjugate is cleaved from the antibody whenit is internalized by the LIV-1-expressing cancer cell (e.g., in theendosomal or, for example by virtue of pH sensitivity or proteasesensitivity, in the lysosomal environment or in the caveolearenvironment).

Typically the ADC comprises a linker region between the therapeuticagent and the anti-LIV-1 antibody. As noted supra, typically, the linkeris cleavable under intracellular conditions, such that cleavage of thelinker releases the therapeutic agent from the antibody in theintracellular environment (e.g., within a lysosome or endosome orcaveolea). The linker can be, e.g., a peptidyl linker that is cleaved byan intracellular peptidase or protease enzyme, including a lysosomal orendosomal protease. Typically, the peptidyl linker is at least two aminoacids long or at least three amino acids long. Cleaving agents caninclude cathepsins B and D and plasmin (see, e.g., Dubowchik and Walker,1999, Pharm. Therapeutics 83:67-123). Most typical are peptidyl linkersthat are cleavable by enzymes that are present in LIV-1-expressingcells. For example, a peptidyl linker that is cleavable by thethiol-dependent protease cathepsin-B, which is highly expressed incancerous tissue, can be used (e.g., a linker comprising a Phe-Leu or aGly-Phe-Leu-Gly (SEQ ID NO:90) peptide). Other such linkers aredescribed, e.g., in U.S. Pat. No. 6,214,345. In specific embodiments,the peptidyl linker cleavable by an intracellular protease comprises aVal-Cit linker or a Phe-Lys dipeptide (see, e.g., U.S. Pat. No.6,214,345, which describes the synthesis of doxorubicin with the Val-Citlinker). One advantage of using intracellular proteolytic release of thetherapeutic agent is that the agent is typically attenuated whenconjugated and the serum stabilities of the conjugates are typicallyhigh.

The cleavable linker can be pH-sensitive, i.e., sensitive to hydrolysisat certain pH values. Typically, the pH-sensitive linker is hydrolyzableunder acidic conditions. For example, an acid-labile linker that ishydrolyzable in the lysosome (e.g., a hydrazone, semicarbazone,thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or thelike) can be used. (See, e.g., U.S. Pat. Nos. 5,122,368; 5,824,805;5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123;Neville et al., 1989, Biol. Chem. 264:14653-14661.) Such linkers arerelatively stable under neutral pH conditions, such as those in theblood, but are unstable at below pH 5.5 or 5.0, the approximate pH ofthe lysosome. In certain embodiments, the hydrolyzable linker is athioether linker (such as, e.g., a thioether attached to the therapeuticagent via an acylhydrazone bond (see, e.g., U.S. Pat. No. 5,622,929)).

Other linkers are cleavable under reducing conditions (e.g., a disulfidelinker). Disulfide linkers include those that can be formed using SATA(N-succinimidyl-S-acetylthioacetate), SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene),SPDB and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res.47:5924-5931; Wawrzynczak et al., In Immunoconjugates: AntibodyConjugates in Radio imagery and Therapy of Cancer (C. W. Vogel ed.,Oxford U. Press, 1987. See also U.S. Pat. No. 4,880,935.)

The linker can also be a malonate linker (Johnson et al., 1995,Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau et al.,1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3′-N-amide analog (Lau etal., 1995, Bioorg-Med-Chem. 3(10):1305-12).

The linker also can be a non-cleavable linker, such as anmaleimido-alkylene- or maleimide-aryl linker that is directly attachedto the therapeutic agent (e.g., a drug). An active drug-linker isreleased by degradation of the antibody.

Typically, the linker is not substantially sensitive to theextracellular environment meaning that no more than about 20%, typicallyno more than about 15%, more typically no more than about 10%, and evenmore typically no more than about 5%, no more than about 3%, or no morethan about 1% of the linkers in a sample of the ADC is cleaved when theADC present in an extracellular environment (e.g., in plasma). Whether alinker is not substantially sensitive to the extracellular environmentcan be determined, for example, by incubating independently with plasmaboth (a) the ADC (the “ADC sample”) and (b) an equal molar amount ofunconjugated antibody or therapeutic agent (the “control sample”) for apredetermined time period (e.g., 2, 4, 8, 16, or 24 hours) and thencomparing the amount of unconjugated antibody or therapeutic agentpresent in the ADC sample with that present in control sample, asmeasured, for example, by high performance liquid chromatography.

The linker can also promote cellular internalization. The linker canpromote cellular internalization when conjugated to the therapeuticagent (i.e., in the milieu of the linker-therapeutic agent moiety of theADC or ADC derivative as described herein). Alternatively, the linkercan promote cellular internalization when conjugated to both thetherapeutic agent and the anti-LIV-1 antibody (i.e., in the milieu ofthe ADC as described herein).

A variety of linkers that can be used with the present compositions aredescribed in WO 2004-010957 and have the form

wherein:

-A- is a stretcher unit;

a is 0 or 1;

each -W- is independently an amino acid unit;

w is independently an integer ranging from 0 to 12;

-Y- is a spacer unit; and

y is 0, 1 or 2.

Representative stretcher units are depicted within the square bracketsof Formulas (Ia) and (Ib; see infra), wherein A-, -W-, -Y-, -D, w and yare as defined above and R¹ is selected from —C₁-C₁₀ alkylene-, —C₃-C₈carbocyclo-, —O—(C₁-C₈ alkyl)-, -arylene-, —C₁-C₁₀ alkylene-arylene-,-arylene-C₁-C₁₀ alkylene-, —C₁-C₁₀ alkylene-(C₃-C₈ carbocyclo)-, —(C₃-C₈carbocyclo)-C₁-C₁₀ alkylene-, —C₃-C₈ heterocyclo-, alkylene-(C₃-C₈heterocyclo)-, —(C₃-C₈ heterocyclo)-C₁-C₁₀ alkylene-, —(CH₂CH₂O)_(r)—,and —(CH₂CH₂O)_(r)—CH₂—; and r is an integer ranging from 1-10. Ab isantibody.

The drug loading is represented by p, the number of drug-linkermolecules per antibody. Depending on the context, p can represent theaverage number of drug-linker molecules per antibody, also referred tothe average drug loading. P ranges from 1 to 20 and is preferably from 1to 8. In some preferred embodiments, when p represents the average drugloading, p ranges from about 2 to about 5. In some embodiments, p isabout 2, about 3, about 4, or about 5. The average number of drugs perantibody in a preparation may be characterized by conventional meanssuch as mass spectroscopy, ELISA assay, and HPLC.

The Amino Acid unit (-W-), if present, links the Stretcher unit (-A-) tothe Spacer unit (-Y-) if the Spacer unit is present, and links theStretcher unit to the cytotoxic or cytostatic agent (Drug unit; D) ifthe spacer unit is absent.

If present, -W_(w)- is preferably a dipeptide, tripeptide, tetrapeptide,pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide,decapeptide, undecapeptide or dodecapeptide unit.

The Spacer unit (-Y-), when present, links an Amino Acid unit to theDrug unit. Spacer units are of two general types: self-immolative andnon self-immolative. A non self-immolative spacer unit is one in whichpart or all of the Spacer unit remains bound to the Drug unit afterenzymatic cleavage of an amino acid unit from the anti-LIV-1antibody-linker-drug conjugate or the drug-linker compound. Examples ofa non self-immolative Spacer unit include a (glycine-glycine) spacerunit and a glycine spacer unit. When an anti-LIV-1 antibody-linker-drugconjugate containing a glycine-glycine spacer unit or a glycine spacerunit undergoes enzymatic cleavage via a tumor-cell associated-protease,a cancer-cell-associated protease or a lymphocyte-associated protease, aglycine-glycine-drug moiety or a glycine-drug moiety is cleaved fromAb-A_(a)-W_(w)-. To liberate the drug, an independent hydrolysisreaction should take place within the target cell to cleave theglycine-drug unit bond.

Alternatively, an anti-LIV-1 antibody drug conjugate containing aself-immolative spacer unit can release the drug (D) without the needfor a separate hydrolysis step. In some of these embodiments, -Y- is ap-aminobenzyl alcohol (PAB) unit that is linked to -W_(w)- via thenitrogen atom of the PAB group, and connected directly to -D via acarbonate, carbamate or ether group. Other examples of self-immolativespacers include aromatic compounds that are electronically equivalent tothe PAB group such as 2-aminoimidazol-5-methanol derivatives (see Hay etal., 1999, Bioorg. Med. Chem. Lett. 9:2237 for examples) and ortho orpara-aminobenzylacetals. Spacers can be used that undergo facilecyclization upon amide bond hydrolysis, such as substituted andunsubstituted 4-aminobutyric acid amides (Rodrigues et al., 1995,Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1] andbicyclo[2.2.2] ring systems (Storm et al., 1972, J. Amer. Chem. Soc.94:5815) and 2-aminophenylpropionic acid amides (Amsberry et al., 1990,J. Org. Chem. 55:5867). Elimination of amine-containing drugs that aresubstituted at the α-position of glycine (Kingsbury, et al., 1984, J.Med. Chem. 27:1447) are also examples of self-immolative spacerstrategies that can be applied to the anti-LIV-1 antibody-linker-drugconjugates. Alternatively, the spacer unit is a branchedbis(hydroxymethyl)styrene (BHMS) unit, which can be used to incorporateadditional drugs.

Useful classes of cytotoxic agents to conjugate to anti-LIV-1 antibodiesinclude, for example, antitubulin agents, DNA minor groove bindingagents, DNA replication inhibitors, chemotherapy sensitizers, or thelike. Other exemplary classes of cytotoxic agents includeanthracyclines, auristatins, camptothecins, duocarmycins, etoposides,maytansinoids and vinca alkaloids. Some exemplary cytotoxic agentsinclude auristatins (e.g., auristatin E, AFP, MMAF, MMAE), DNA minorgroove binders (e.g., enediynes and lexitropsins), duocarmycins, taxanes(e.g., paclitaxel and docetaxel), vinca alkaloids, doxorubicin,morpholino-doxorubicin, and cyanomorpholino-doxorubicin.

The cytotoxic agent can be a chemotherapeutic such as, for example,doxorubicin, paclitaxel, melphalan, vinca alkaloids, methotrexate,mitomycin C or etoposide. The agent can also be a CC-1065 analogue,calicheamicin, maytansine, an analog of dolastatin 10, rhizoxin, orpalytoxin.

The cytotoxic agent can also be an auristatin. The auristatin can be anauristatin E derivative is, e.g., an ester formed between auristatin Eand a keto acid. For example, auristatin E can be reacted withparaacetyl benzoic acid or benzoylvaleric acid to produce AEB and AEVB,respectively. Other typical auristatins include AFP, MMAF, and MMAE. Thesynthesis and structure of various auristatins are described in, forexample, US 2005-0238649 and US2006-0074008.

The cytotoxic agent can be a DNA minor groove binding agent. (See, e.g.,U.S. Pat. No. 6,130,237.) For example, the minor groove binding agentcan be a CBI compound or an enediyne (e.g., calicheamicin).

The cytotoxic or cytostatic agent can be an anti-tubulin agent. Examplesof anti-tubulin agents include taxanes (e.g., Taxol® (paclitaxel),Taxotere® (docetaxel)), T67 (Tularik), vinca alkyloids (e.g.,vincristine, vinblastine, vindesine, and vinorelbine), and auristatins(e.g., auristatin E, AFP, MMAF, MMAE, AEB, AEVB). (Exemplary auristatinsare shown below in formulae III-XII. Other suitable antitubulin agentsinclude, for example, baccatin derivatives, taxane analogs (e.g.,epothilone A and B), nocodazole, colchicine and colcimid, estramustine,cryptophysins, cemadotin, maytansinoids, combretastatins,discodermolide, and eleutherobin.

The cytotoxic agent can be a maytansinoid, another group of anti-tubulinagents. For example, the maytansinoid can be maytansine or a maytansinecontaining drug linker such as DM-1 or DM-4 (ImmunoGen, Inc.; see alsoChari et al., 1992, Cancer Res. 52:127-131).

Exemplary antibody drug conjugates include vcMMAE and mcMMAF antibodydrug conjugates as follows wherein p and Ab are as previously describedherein:

or a pharmaceutically acceptable salt thereof.VI. Other Antibodies to LIV-1

As well as humanized forms of the BR2-14a and BR2-22a antibodiesdiscussed above, other antibodies binding to an extracellular domain ofLIV-1 can be used in some of the methods of the invention, particularlythe treatment of triple negative breast cancers. A collection of mouseantibodies to LIV-1 is described in US20080175839. These antibodiesinclude 1.1F10, 1.7A4, BR2-10b, BR2-11a, BR2-13a, BR2-14a, BR2-15a,BR2-16a, BR2-17a, BR2-18a, BR2-19a, BR2-20a, BR2-21a, BR2-22a, BR2-23a,BR2-24a, and BR2-25a, of which BR2-19a produced by the hybridoma ATCCAccession No. PTA-5706 or BR2-23a produced by the hybridoma ATCCAccession No. PTA-5707 in addition to BR2-14a and BR2-22a are preferred.Humanized, chimeric or veneered forms of these antibodies can be made byconventional methods summarized below.

Other antibodies to LIV-1 can be made de novo by immunizing with LIV-1or one or more extracellular domains thereof. The production of othernon-human monoclonal antibodies, e.g., murine, guinea pig, primate,rabbit or rat, against an immunogen can be performed by as described byHarlow & Lane, Antibodies, A Laboratory Manual (CSHP NY, 1988)(incorporated by reference for all purposes). Such an immunogen can beobtained from a natural source, by peptide synthesis or by recombinantexpression.

Humanized, chimeric or veneered forms of non-human antibodies can bemade. General methodology for producing humanized antibodies isdescribed by Queen, U.S. Pat. Nos. 5,530,101 and 5,585,089; Winter, U.S.Pat. No. 5,225,539; Carter, U.S. Pat. No. 6,407,213; Adair, U.S. Pat.No. 5,859,205; and Foote, U.S. Pat. No. 6,881,557). A chimeric antibodyis an antibody in which the mature variable regions of light and heavychains of a non-human antibody (e.g., a mouse) are combined with humanlight and heavy chain constant regions. Such antibodies substantially orentirely retain the binding specificity of the mouse antibody, and areabout two-thirds human sequence. A veneered antibody is a type ofhumanized antibody that retains some and usually all of the CDRs andsome of the non-human variable region framework residues of a non-humanantibody but replaces other variable region framework residues that maycontribute to B- or T-cell epitopes, for example exposed residues(Padlan, Mol. Immunol. 28:489, 1991) with residues from thecorresponding positions of a human antibody sequence. The result is anantibody in which the CDRs are entirely or substantially from anon-human antibody and the variable region frameworks of the non-humanantibody are made more human-like by the substitutions.

Human antibodies against LIV-1 can be provided by a variety oftechniques described below. Methods for producing human antibodiesinclude the trioma method of Oestberg et al., Hybridoma 2:361-367(1983); Oestberg, U.S. Pat. No. 4,634,664; and Engleman et al., U.S.Pat. No. 4,634,666; use of transgenic mice including humanimmunoglobulin genes (see, e.g., Lonberg et al., WO93/12227 (1993); U.S.Pat. No. 5,877,397, U.S. Pat. No. 5,874,299, U.S. Pat. No. 5,814,318,U.S. Pat. No. 5,789,650, U.S. Pat. No. 5,770,429, U.S. Pat. No.5,661,016, U.S. Pat. No. 5,633,425, U.S. Pat. No. 5,625,126, U.S. Pat.No. 5,569,825, U.S. Pat. No. 5,545,806, Nature 148, 1547-1553 (1994),Nature Biotechnology 14, 826 (1996), Kucherlapati, WO 91/10741 (1991)and phage display methods (see, e.g. Dower et al., WO 91/17271 andMcCafferty et al., WO 92/01047, U.S. Pat. No. 5,877,218, U.S. Pat. No.5,871,907, U.S. Pat. No. 5,858,657, U.S. Pat. No. 5,837,242, U.S. Pat.No. 5,733,743 and U.S. Pat. No. 5,565,332.

Any of the antibodies can be selected to have the same or overlappingepitope specificity as an exemplar antibody, such as the BR2-14aantibody, by a competitive binding assay or otherwise.

VII. Therapeutic Applications

The humanized antibodies of the invention, alone or as LIV-1 antibodydrug conjugates thereof, can be used to treat cancer. Some such cancersshow detectable levels of LIV-1 measured at either the protein (e.g., byimmunoassay using one of the exemplified antibodies) or mRNA level. Somesuch cancers show elevated levels of LIV-1 relative to noncanceroustissue of the same type, preferably from the same patient. An exemplarylevel of LIV-1 on cancer cells amenable to treatment is 5000-150000LIV-1 molecules per cell, although higher or lower levels can betreated. Optionally, a level of LIV-1 in a cancer is measured beforeperforming treatment.

Examples of cancers associated with LIV-1 expression and amenable totreatment include breast cancer, prostate cancer, ovarian cancer,endometrial cancer, cervical, liver, gastric, kidney, and squamous cellcarcinomas (e.g., bladder, head, neck and lung), skin cancers, e.g.,melanoma, small lung cell carcinoma or lung carcinoid. The treatment canbe applied to patients having primary or metastatic tumors of thesekinds. The treatment can also be applied to patients who are refractoryto conventional treatments (e.g., hormones, tamoxifen, herceptin), orwho have relapsed following a response to such treatments. The methodscan also be used on triple negative breast cancers. A triple negativebreast cancer is a term of art for a cancer lacking detectable estrogenand progesterone receptors and lacking overexpression of HER2/neu whenstained with an antibody to any of these receptors, such as described inthe examples. Staining can be performed relative to an irrelevantcontrol antibody and lack of expression shown from a background level ofstraining the same or similar to that of the control within experimentalerror. Likewise lack of overexpression is shown by staining at the sameor similar level within experimental error of noncancerous breasttissue, preferably obtained from the same patient. Alternatively oradditionally, triple native breast cancers are characterized by lack ofresponsiveness to hormones interacting with these receptors, aggressivebehavior and a distinct pattern of metastasis.

hLIV14 antibodies can be used to treat cancers that express LIV-1. Inone embodiment, an hLIV14 antibody is used treat a subject with aLIV-1-expressing breast cancer. In another embodiment, an hLIV14antibody is used treat a subject with a LIV-1-expressing prostatecancer. In another embodiment, an hLIV14 antibody is used treat asubject with a LIV-1-expressing melanoma. In another embodiment, anhLIV14 antibody is used treat a subject with a LIV-1-expressing ovariancancer. In another embodiment, an hLIV14 antibody is used treat asubject with a LIV-1-expressing endometrial cancer. In anotherembodiment, an hLIV14 antibody is used treat a subject with aLIV-1-expressing cervical cancer. In another embodiment, an hLIV14antibody is used treat a subject with a LIV-1-expressing liver cancer.In another embodiment, an hLIV14 antibody is used treat a subject with aLIV-1-expressing gastric cancer. In another embodiment, an hLIV14antibody is used treat a subject with a LIV-1-expressing kidney cancer.In another embodiment, an hLIV14 antibody is used treat a subject with aLIV-1-expressing squamous cell carcinomas (e.g., bladder, head, neck andlung cancer). In another embodiment, an hLIV14 antibody is used treat asubject with a LIV-1-expressing breast cancer. In another embodiment, anhLIV14 antibody is used treat a subject with a LIV-1-expressing skincancer. In another embodiment, an hLIV14 antibody is used treat asubject with a LIV-1-expressing small lung cell carcinoma or lungcarcinoid. hLIV22 antibodies can be used to treat cancers that expressLIV-1. In one embodiment, an hLIV22 antibody is used treat a subjectwith a LIV-1-expressing breast cancer. In another embodiment, an hLIV22antibody is used treat a subject with a LIV-1-expressing prostatecancer. In another embodiment, an hLIV22 antibody is used treat asubject with a LIV-1-expressing melanoma. In another embodiment, anhLIV22 antibody is used treat a subject with a LIV-1-expressing ovariancancer. In another embodiment, an hLIV22 antibody is used treat asubject with a LIV-1-expressing endometrial cancer. In anotherembodiment, an hLIV22 antibody is used treat a subject with aLIV-1-expressing cervical cancer. In another embodiment, an hLIV22antibody is used treat a subject with a LIV-1-expressing liver cancer.In another embodiment, an hLIV22 antibody is used treat a subject with aLIV-1-expressing gastric cancer. In another embodiment, an hLIV22antibody is used treat a subject with a LIV-1-expressing kidney cancer.In another embodiment, an hLIV22 antibody is used treat a subject with aLIV-1-expressing squamous cell carcinomas (e.g., bladder, head, neck andlung cancer). In another embodiment, an hLIV22 antibody is used treat asubject with a LIV-1-expressing breast cancer. In another embodiment, anhLIV22 antibody is used treat a subject with a LIV-1-expressing skincancer. In another embodiment, an hLIV22 antibody is used treat asubject with a LIV-1-expressing small lung cell carcinoma or lungcarcinoid. This application provides the first disclosure that LIV-1protein is expressed on the surface of melanoma cells. Thus, antibodiesthat bind to LIV-1 can be used to treat patients that are afflicted withmelanomas that express LIV-1. Such antibodies include antibodiesdisclosed herein, e.g., hLIV14 and hLIV22, but are not limited to theantibodies disclosed herein.

Humanized antibodies, alone or as conjugates thereof, are administeredin an effective regime meaning a dosage, route of administration andfrequency of administration that delays the onset, reduces the severity,inhibits further deterioration, and/or ameliorates at least one sign orsymptom of cancer. If a patient is already suffering from cancer, theregime can be referred to as a therapeutically effective regime. If thepatient is at elevated risk of the cancer relative to the generalpopulation but is not yet experiencing symptoms, the regime can bereferred to as a prophylactically effective regime. In some instances,therapeutic or prophylactic efficacy can be observed in an individualpatient relative to historical controls or past experience in the samepatient. In other instances, therapeutic or prophylactic efficacy can bedemonstrated in a preclinical or clinical trial in a population oftreated patients relative to a control population of untreated patients.

Exemplary dosages for a monoclonal antibody are 0.1 mg/kg to 50 mg/kg ofthe patient's body weight, more typically 1 mg/kg to 30 mg/kg, 1 mg/kgto 20 mg/kg, 1 mg/kg to 15 mg/kg, 1 mg/kg to 12 mg/kg, or 1 mg/kg to 10mg/kg1, or 2 mg/kg to 30 mg/kg, 2 mg/kg to 20 mg/kg, 2 mg/kg to 15mg/kg, 2 mg/kg to 12 mg/kg, or 2 mg/kg to 10 mg/kg, or 3 mg/kg to 30mg/kg, 3 mg/kg to 20 mg/kg, 3 mg/kg to 15 mg/kg, 3 mg/kg to 12 mg/kg, or3 mg/kg to 10 mg/kg. Exemplary dosages for a monoclonal antibody orantibody drug conjugates thereof are 1 mg/kg to 7.5 mg/kg, or 2 mg/kg to7.5 mg/kg or 3 mg/kg to 7.5 mg/kg of the subject's body weight, or0.1-20, or 0.5-5 mg/kg body weight (e.g., 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9or 10 mg/kg) or 10-1500 or 200-1500 mg as a fixed dosage. In somemethods, the patient is administered a dose of at least 1.5 mg/kg, atleast 2 mg/kg or at least 3 mg/kg, administered once every three weeksor greater. The dosage depends on the frequency of administration,condition of the patient and response to prior treatment, if any,whether the treatment is prophylactic or therapeutic and whether thedisorder is acute or chronic, among other factors.

Administration can be parenteral, intravenous, oral, subcutaneous,intra-arterial, intracranial, intrathecal, intraperitoneal, topical,intranasal or intramuscular. Administration can also be localizeddirectly into a tumor. Administration into the systemic circulation byintravenous or subcutaneous administration is preferred. Intravenousadministration can be, for example, by infusion over a period such as30-90 min or by a single bolus injection.

The frequency of administration depends on the half-life of the antibodyor conjugate in the circulation, the condition of the patient and theroute of administration among other factors. The frequency can be daily,weekly, monthly, quarterly, or at irregular intervals in response tochanges in the patient's condition or progression of the cancer beingtreated. An exemplary frequency for intravenous administration isbetween twice a week and quarterly over a continuous course oftreatment, although more or less frequent dosing is also possible. Otherexemplary frequencies for intravenous administration are between weeklyor three out of every four weeks over a continuous course of treatment,although more or less frequent dosing is also possible. For subcutaneousadministration, an exemplary dosing frequency is daily to monthly,although more or less frequent dosing is also possible.

The number of dosages administered depends on the nature of the cancer(e.g., whether presenting acute or chronic symptoms) and the response ofthe disorder to the treatment. For acute disorders or acuteexacerbations of a chronic disorder between 1 and 10 doses are oftensufficient. Sometimes a single bolus dose, optionally in divided form,is sufficient for an acute disorder or acute exacerbation of a chronicdisorder. Treatment can be repeated for recurrence of an acute disorderor acute exacerbation. For chronic disorders, an antibody can beadministered at regular intervals, e.g., weekly, fortnightly, monthly,quarterly, every six months for at least 1, 5 or 10 years, or the lifeof the patient.

Pharmaceutical compositions for parenteral administration are preferablysterile and substantially isotonic and manufactured under GMPconditions. Pharmaceutical compositions can be provided in unit dosageform (i.e., the dosage for a single administration). Pharmaceuticalcompositions can be formulated using one or more physiologicallyacceptable carriers, diluents, excipients or auxiliaries. Theformulation depends on the route of administration chosen. Forinjection, antibodies can be formulated in aqueous solutions, preferablyin physiologically compatible buffers such as Hank's solution, Ringer'ssolution, or physiological saline or acetate buffer (to reducediscomfort at the site of injection). The solution can containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively antibodies can be in lyophilized form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use. The concentration of antibody in a liquid formulation can bee.g., 1-100 mg/ml, such as 10 mg/ml.

Treatment with antibodies of the invention can be combined withchemotherapy, radiation, stem cell treatment, surgery other treatmentseffective against the disorder being treated. Useful classes of otheragents that can be administered with humanized antibodies to LIV-1include, for example, antibodies to other receptors expressed oncancerous cells, antitubulin agents (e.g., auristatins), DNA minorgroove binders, DNA replication inhibitors, alkylating agents (e.g.,platinum complexes such as cis-platin, mono(platinum), bis(platinum) andtri-nuclear platinum complexes and carboplatin), anthracyclines,antibiotics, antifolates, antimetabolites, chemotherapy sensitizers,duocarmycins, etoposides, fluorinated pyrimidines, ionophores,lexitropsins, nitrosoureas, platinols, pre-forming compounds, purineantimetabolites, puromycins, radiation sensitizers, steroids, taxanes,topoisomerase inhibitors, vinca alkaloids, and the like.

Treatment with the humanized anti-LIV-1 antibody, optionally incombination with any of the other agents or regimes described abovealone or as an antibody drug conjugate, can increase the medianprogression-free survival or overall survival time of patients withtumors (e.g., breast, prostate, melanoma), especially when relapsed orrefractory, by at least 30% or 40% but preferably 50%, 60% to 70% oreven 100% or longer, compared to the same treatment (e.g., chemotherapy)but without an anti-LIV-1 antibody alone or as a conjugate. In additionor alternatively, treatment (e.g., standard chemotherapy) including theanti-LIV-1 antibody alone or as a conjugate can increase the completeresponse rate, partial response rate, or objective response rate(complete+partial) of patients with tumors by at least 30% or 40% butpreferably 50%, 60% to 70% or even 100% compared to the same treatment(e.g., chemotherapy) but without the anti-LIV-1 antibody.

Typically, in a clinical trial (e.g., a phase II, phase II/III or phaseIII trial), the aforementioned increases in median progression-freesurvival and/or response rate of the patients treated with standardtherapy plus the humanized anti-LIV-1 antibody, relative to the controlgroup of patients receiving standard therapy alone (or plus placebo),are statistically significant, for example at the p=0.05 or 0.01 or even0.001 level. The complete and partial response rates are determined byobjective criteria commonly used in clinical trials for cancer, e.g., aslisted or accepted by the National Cancer Institute and/or Food and DrugAdministration.

VIII. Other Applications

The anti-LIV-1 humanized antibodies can be used for detecting LIV-1 inthe context of clinical diagnosis or treatment or in research.Expression of LIV-1 on a cancer provides an indication that the canceris amenable to treatment with the antibodies of the present invention.The antibodies can also be sold as research reagents for laboratoryresearch in detecting cells bearing LIV-1 and their response to variousstimuli. In such uses, monoclonal antibodies can be labeled withfluorescent molecules, spin-labeled molecules, enzymes or radioisotypes,and can be provided in the form of kit with all the necessary reagentsto perform the assay for LIV-1. The antibodies described herein,BR2-14a, BR2-22a and humanized versions thereof, e.g., hLIV14 andhLIV22, can be used to detect LIV-1 protein expression and determinewhether a cancer is amenable to treatment with LIV-1 ADCs. As anexample, BR2-14a, BR2-22a and humanized versions thereof, e.g., hLIV14and hLIV22 can be used to detect LIV-1 expression on breast cancercells, melanoma cells, cervical cancer cells, or prostate cancer cells.The antibodies can also be used to purify LIV-1, e.g., by affinitychromatography.

IX. Cynomolgus Monkey LIV-1

The invention further provides an amino acid sequence for LIV-1 (CYLIV-1) from cynomolgus monkeys at SEQ ID NO:85 with or without a signalpeptide, which occupies approximately residues 1-28 of SEQ ID NO:85, aswell as nucleic acids that encode that amino acid sequences. Variantsdiffering by up to 1, 2, 3, 4, or 5 substitutions, deletions orinsertions are also included provided CY variants do not include anatural human LIV-1 sequence. Analogous to human LIV-1, reference toCY-LIV-1 means at least an extracellular domain of the protein andusually the complete protein other than a cleavable signal peptide(amino acids 1-28). The invention further provides antibodies thatspecifically bind to SEQ ID NO:85 with or without specifically bindingto human LIV-1 (i.e., binding to human LIV-1 at level of negativecontrol irrelevant antibody). The invention further provides antibodiesthat preferentially bind CY-LIV-1 over human LIV-1 and vice versa.Preferential binding means an association higher beyond experimentalerror and preferably at least 2, 3 or 4 fold higher. The inventionfurther provides antibodies that show the same binding profile to humanand CY LIV-1 within experimental error as any of the exemplifiedantibodies described below. The invention further provides methods ofanalyzing binding of an antibody to CY LIV-1. Such methods involvecontacting an antibody with CY LIV-1, determining whether the antibodyspecifically binds to CY LIV-1 and optionally determining a measure ofbinding strength, such as an association constant.

All patent filings, website, other publications, accession numbers andthe like cited above or below are incorporated by reference in theirentirety for all purposes to the same extent as if each individual itemwere specifically and individually indicated to be so incorporated byreference. If different versions of a sequence are associated with anaccession number at different times, the version associated with theaccession number at the effective filing date of this application ismeant. The effective filing date means the earlier of the actual filingdate or filing date of a priority application referring to the accessionnumber if applicable. Likewise if different versions of a publication,website or the like are published at different times, the version mostrecently published at the effective filing date of the application ismeant unless otherwise indicated. Any feature, step, element,embodiment, or aspect of the invention can be used in combination withany other unless specifically indicated otherwise. Although the presentinvention has been described in some detail by way of illustration andexample for purposes of clarity and understanding, it will be apparentthat certain changes and modifications may be practiced within the scopeof the appended claims.

EXAMPLES

I. Humanization of BR2-14a

Materials

Cell lines described in the following examples were maintained inculture according to the conditions specified by the American TypeCulture Collection (ATCC), the National Cancer Institute (NCI) or theDeutsche Sammlung von Mikroorganismen and Zellkulturen GmbH,Braunschweig, Germany (DMSZ). Cell culture reagents were obtained fromInvitrogen Corp. (Carlsbad, Calif.) or other suppliers.

Methodologies:

Saturation Binding Assays

1×10⁵ antigen expressing cells (either MCF7 cells (ATCC) expressinghuman LIV-1, a transfected CHO cell line expressing human LIV-1 or atransfected CHO cell line expressing cyno LIV-1) were aliquoted per wellof a 96-well v-bottom plates. AlexaFluor-647 labeled murine LIV-1 mAb,e.g., BR2-14a, was added in concentrations ranging from 0.66 pM to 690nM and incubated on ice for 30 minutes. Cells were pelleted and washed3× with PBS/BSA. The cells were then pelleted and resuspended in 125 μLof PBS/BSA. Fluorescence was analyzed by flow cytometry, using percentof saturated fluorescent signal to determine percent bound and tosubsequently calculate apparent Kd.

Competition Binding Assays

1×10⁵ CHO cells expressing recombinant human LIV-1 in PBS/BSA werealiquoted into each well of a 96-well v-bottom plates on ice. The cellswere incubated for 1 hour with 5 nM AlexaFluor-647 (AF) labeled parentalmurine LIV-1 mAb and increasing concentrations (from 0.038 nM to 600 nM)of unlabeled humanized LIV-1 mAb, combinations of humanized light chainsLA-LF and humanized heavy chains HA-HE. Cells were pelleted and washed 3times with PBS/BSA. The cells were pelleted and resuspended in 125 μL ofPBS/BSA. Fluorescence was analyzed by flow cytometry, using percent ofsaturated fluorescent signal to determine percent labeled murine LIV-1mAb bound and to subsequently extrapolate the EC50 by fitting the datato a sigmoidal dose-response curve with variable slope.

1×10⁵ MCF7 cells expressing LIV-1 in PBS/BSA were aliquoted in each wellof a 96-well v-bottom plates on ice. The cells were incubated for 1 hourwith 5 nM AlexaFluor-647 labeled murine LIV-1 mAb and increasingconcentrations (from 0.038 nM to 600 nM) of unlabeled humanized LIV-1mAb, combinations of humanized light chains LA-LF and humanized heavychains HA-HE. Cells were pelleted and washed 3 times with PBS. The cellswere pelleted and resuspended in 125 μL of PBS/BSA. Fluorescence wasanalyzed by flow cytometery, using percent of saturated fluorescentsignal to determine percent labeled murine LIV-1 mAb bound and tosubsequently extrapolate the EC50 by fitting the data to a sigmoidaldose-response curve with variable slope.

1×10⁵ CHO cells expressing recombinant cyno LIV-1 in PBS were aliquotedin each well of a 96-well v-bottom plates on ice. The cells wereincubated for 1 hour with 5 nM AlexaFluor-647 labeled murine LIV-1 mAband increasing concentrations (from 0.038 nM to 600 nM) of unlabeledhumanized LIV-1 mAb, combinations of humanized light chains LA-LF andhumanized heavy chains HA-HE. Cells were pelleted and washed 3 timeswith PBS. The cells were pelleted and resuspended in 125 μL of PBS/BSA.Fluorescence was analyzed by flow cytometry, using percent of saturatedfluorescent signal to determine percent labeled murine LIV-1 mAb boundand to subsequently extrapolate the EC50 by fitting the data to asigmoidal dose-response curve with variable slope.

Quantitative Flow Cytometric Analysis

Quantification of LIV-1 copy number on the cell surfaces was determinedusing murine LIV-1 mAb as primary antibody and the DAKO QiFiKit flowcytometric indirect assay as described by the manufacturer (DAKO A/S,Glostrup, Denmark) and evaluated with a Becton Dickinson FACS®can flowcytometer.

Cytotoxicity Assay

Tumor cells were incubated with LIV-1 antibody drug conjugates for96-144 hours at 37° C. A non-binding (H00) ADC was used as a negativecontrol. Cell viability was measured by resazurin (Sigma) at the finalconcentration of 50 μM. Cells were incubated for four to six hours at37°. Fluorescent signal was measured on a Fusion HT fluorescent platereader (Perkin Elmer, Waltham, Mass.). Results are reported as IC₅₀, theconcentration of compound needed to yield a 50% reduction in viabilitycompared to vehicle-treated cells (control=100%).

Production of Antibody Drug Conjugates

Antibody drug conjugates of the LIV-1 antibodies were prepared asdescribed in US20050238649. The drug linkers vcMMAE (also referred to as1006) and mcMMAF (referred to as 1269) are both described inUS20050238649. Preparation of cysteine mutants of IgG1 antibodies isgenerally described in US20100158919. US20050238649 and US20100158919are herein incorporated by reference for all purposes.

Production of Non-Fucosylated Anti-LIV-1 mAb

A CHO DG44 cell line producing the humanized IgG1 anti-LIV-1 monoclonalantibody, HBLB mAb (hLIV-14), was cultured at 3.0×10⁵ cells per mL in 30mL of CHO culture media at 37°, 5% CO₂ and shaking at 100 RPM in a 125mL shake flask. Media was supplemented with insulin like growth factor(IGF), penicillin, streptomycin and 65 μM 2-fluorofucose peracetate(SGD-2084) (see US20090317869). Cultures were fed on day 3 with 2%volume of feed media. On day four, the culture was split 1:4 into freshculture media. Cultures were fed with a 6% volume of production feedmedia on days 5, 7, 9 and 10. Conditioned media was collected on day 13by passing the culture through a 0.2 μm filter. Antibody purificationwas performed by applying the conditioned media to a protein A columnpre-equilibrated with 1× phosphate buffered saline (PBS), pH 7.4.

After washing column with 20 column volumes of 1×PBS, antibodies wereeluted with 5 column volumes of Immunopure IgG elution buffer (PierceBiotechnology, Rockford, Ill.). A 10% volume of 1M Tris pH 8.0 was addedto eluted fraction. Sample was dialyzed overnight into 1×PBS.

Antibody-Dependent Cellular Cytotoxicity (ADCC)

ADCC activity was measured using the standard ⁵¹Cr-release assay.Briefly, the MCF-7 target tumor cells were labeled with 100 μCiNa⁵¹CrO₄, washed, and pre-incubated with test antibodies prior toaddition of effector (natural killer, NK) cells. NK (CD16⁺ CD56⁺) cellswere prepared from non-adherent peripheral blood mononuclear cells(PBMCs) obtained from normal FcγRIIIA 158V/V donors (Lifeblood, Memphis,Tenn.) with immunomagnetic beads (EasySep, StemCell Technologies,Vancouver, BC, Canada). Viable NK cells were added to target cells at aneffector to target cell ratio of 10:1. A human IgG1κ (Ancell, Bayport,Minn.) was used as negative control in this assay. After 4 hours ofincubation, supernatants were collected and dried overnight on Lumaplates. Gamma radiation emitted from lysed MCF-7 cells was then detectedusing the TopCount Microplate Scintillation and Luminescence Counter(Perkin Elmer, Waltham, Mass.). ADCC activity is reported as % specificlysis.

In Vivo Activity Study

Nude (nu/nu) mice (7-8 animals/group) were implanted with tumor cellsgrown in culture: MCF-7 from NCI (5×10⁶ cells in 25% matrigel), PC3 fromATCC (2.5×10⁶ cells in 25% matrigel), and PC3 from DSMZ (5×10⁵ in 25%matrigel). For in vivo growth of MCF-7 cells, female mice also receivedestrogen supplementation by implanting a slow-release estrogen pellet(90 day release). Dosing with either chimeric or humanized LIV-1 ADC ornonbinding control ADC (3 mg/kg) started when tumors reached 100 mm³(q4d×4 intraperitoneal injections). Tumor volumes were monitored usingcalipers and animals were euthanized when tumor volume reached ˜800 mm³.Median tumor volume plots were continued for each group until one ormore animals were euthanized. All animal procedures were performed undera protocol approved by the Institutional Animal Care and Use Committeein a facility accredited by the Association for Assessment andAccreditation of Laboratory Animal Care.

LIV-1 Immunohistochemical (MC) Staining

Method

Tumor microarrays (TMAs) and individual tumor samples were obtained fromcommercial sources. Tissue microarrays from normal or tumor formalinfixed and paraffin embedded (FFPE) tissues were purchased either from USBiomax Inc. or Cybrdi. A frozen array was purchased from BioChain.Single sections were purchased from NDRI, Asterand, Tissue Solution orCHTN. A set of 25 paraffin-embedded samples of metastatic hormonerefractory prostate cancer (corresponding bone and soft tissuemetastatic sites) was provided by Dr. R. Vessella, University ofWashington, Genitourinary Cancer Department. All samples were processedon Bond-Max™ auto-stainer (Leica).

IHC Staining of FFPE Tissues:

FFPE slides or TMAs sectioned on glass slides were de-paraffinized usingBond™ Dewax solution (Leica, cat # AR9222) at 72° C. and rehydrated.Antigen retrieval was performed using EDTA based Bond™ Epitope RetrievalSolution 2 (Leica, cat # AR9640) for 20 min at 95-100° C. beforeincubation with the primary murine LIV-1 mAb (1-2 μg/ml for 30-45minutes at 25° C.). Isotype-matched murine IgG1 (Sigma; cat # M5284) wasused as negative control for background staining. For automated IHCstaining we used either a Refine DAB kit or an alkaline phosphatasebased detection kit: Bond™ Polymer AP Red Detection kit (Leica, cat #DS9305). Slides were incubated with murine monoclonal primary antibodiesagainst murine LIV-1 mAb for 45 min at 1 μg/ml with a preliminary 30 minprotein block (DAKO cat #X0909). After chromogen development, sectionswere counterstained with hematoxylin and coverslipped. Slides wereevaluated and scored by a pathologist and images were taken using aZeiss Axiovert 200M microscope (Carl Zeiss, Inc., Thornwood, N.Y.).

IHC of Frozen Tissues:

5 μm sections of frozen/OCT samples were acetone fixed for 10 min., airdried for 30 min, and pretreated 20 min with 1× Morphosave at roomtemperature. The slides were loaded into Bond-Max™ auto-stainer (Leica)and stained for 45 min with primary antibody with preliminary 30 minprotein block (DAKO cat# X0909). Mouse IgG1 (BD Pharmingen cat #550878)was used as negative control. For detection we used DAB-based BondPolymer Refine kit (Leica, cat # DS9800). After chromogen development,sections were counterstained with hematoxylin and coverslipped. Slideswere evaluated and scored by pathologist.

Results

1. Binding of Mouse Antibody

The K_(D) for the murine LIV-1 monoclonal antibody BR2-14a antibody(US2004141983) was determined for human LIV-1 expressed as an endogenousprotein in a human breast cancer cell line or as a recombinant proteinin a CHO cell line. The K_(D) for the murine LIV-1 antibody BR2-14a wasalso determined for cyno LIV-1 expressed as a recombinant protein in aCHO cell line. MCF7 is a human breast cancer cell line. 293F is a humanembryonic kidney cell line. Table 1 shows that the antibody had about5-fold lower dissociation constant for non-recombinant LIV-1 expressedfrom a human cell line than recombinant LIV-1, whether human (hLIV-1) orfrom cynomolgus monkeys (cyLIV-1).

TABLE 1 Cell line Antigen Kd (nM) MCF-7 (ATCC) hLIV-1 2.4 293F (hLIV-1)hLIV-1 2.7 CHO (hLIV-1) hLIV-1 12.5 CHO (cyLIV-1) cLIV-1 14.02. Design and Testing of Humanized Antibodies

The starting point or donor antibody for humanization in this Example isthe mouse antibody BR2-14a produced by the hybridoma having ATCCAccession No. PTA-5705A and described in US2004141983. Suitable humanacceptor sequences are genomic sequences provided by VH1-02 and JH5 forthe heavy chain and by VK2-30 and Jk4 for the light chain. The humanacceptor sequences show 68 and 85 percentage identity to the donorsequences in the variable region frameworks. The light chain CDRs of thehuman acceptor sequences are of the same canonical type as the CDRs ofthe donor sequences. In contrast, the heavy chain CDRs of the humanacceptor sequences differed in their canonical type (the germline was1-3 versus 1-2 for the murine donor).

Alignment of the donor sequences identified eleven positions in theheavy chain (H27, H28, H29, H30, H48, H66, H67, H71, H76, H93 and H94)and five positions in the light chain (L36, L37, L45, L46 and L39) atwhich the human acceptor sequence differed from the donor sequence andthat may affect antibody binding as a result of contacting antigendirectly, affecting conformation of CDRs or affecting packing betweenheavy and light chains. Five humanized heavy chains and six humanizedlight chains were made incorporating back mutations at differentpermutations of these positions (FIG. 1 (sequence alignment) and Table2).

TABLE 2 Backmutations VH exon acceptor V_(H) variant sequence donorframework residues hV_(H) A VH1-02 none hV_(H) B VH1-02 H29, H30, H76hV_(H) C VH1-02 H66, H67, H71 hV_(H) D VH1-02 H27, H93, H94 hV_(H) EVH1-02 H27, H28, H29, H30, H48, H76, H66, H67, H71, H93, H94 VL exonacceptor V_(L) variant sequence donor framework residues hV_(K) A VK2-30none hV_(K) B VK2-30 L36 hV_(K) C VK2-30 L37 hV_(K) D VK2-30 L45 hV_(K)E VK2-30 L46 hV_(K) F VK2-30 L36, L37, L39, L45, L46

Humanized antibodies were then expressed representing every permutationof these chains (30 possibilities) of the humanized heavy and lightchains. The binding curves for recombinant human LIV-1 expressed fromCHO cells are shown in FIG. 2. The EC50's are summarized in the Table 3below.

TABLE 3 EC₅₀s for humanized LIV-1 mAb antibodies, derived from BR2-14a,on human LIV-1 expressed in CHO cells Ab EC50 (μg/mL) HALA DNB HALB 37.8HALC 25.5 HALD  4.9 HALE DNB HALF  8.8 HBLA 19.9 HBLB  0.3 HBLC 44.0HBLD 17.4 HBLE DNB HBLF  0.7 HCLA DNB HCLB  1.8 HCLC DNB HCLD 66.6 HCLEDNB HCLF  1.3 HDLA DNB HDLB  2.3 HDLC DNB HDLD 67.9 HDLE DNB HDLF  1.4HELA 12.5 HELB 173.3  HELC DNB HELD 24.2 HELE  0.3 HELF  1.5 DNB means“did not bind”

These data indicate considerable variation of EC50 between the 30humanized antibodies tested with HBLB and HELE showing at least two foldbetter binding that the next humanized antibody HBLF and larger marginsover most of the humanized antibodies. The binding curves of FIG. 2 showthat both HBLB and HELE had stronger binding than the original mouseantibody.

The HBLB antibody was selected as the best of the humanized antibodiesbecause it has (together with HELE) the strongest binding but has fewerbackmutations versus HELE, there being four back mutations in HBLB andtwelve in HELE.

The EC50s for the humanized LIV-1 mAb which bound human LIV-1 expressedon CHO cells were determined for human LIV-1 expressed as a nativeprotein in an MCF7 cell line (FIG. 3). Again, LIV-1 mAb HBLB and HELEwere determined to be the tightest binders.

The Kd for HBLB to human LIV-1 on the MCF7 cell line was determined fromthe average of several saturation binding curves as 1.5 nM whereas thatfor the mouse antibody is 2.9 nM. In other words, the HBLB antibody hasabout twice the affinity for native human LIV-1 as the mouse antibody.The saturation binding curve shown in FIG. 4 is a representativeexample.

Two forms of the HBLB were compared for binding to human LIV-1recombinantly expressed from CHO cells. One form was expressed withwildtype human IgG1 and kappa constant regions. The other form was thesame except for an S239C mutation (EU numbering) in the IgG1 heavy chain(referred to as LIV-14d or HBLB S239C), which reduces binding of theantibody to Fc gamma receptors. The binding curves and EC50's of theseantibodies compared with the mouse donor antibody are shown in FIG. 5.The EC50's of both forms of HBLB were similar to one another (within theerror of the study), and both were stronger than the mouse antibody.

The EC50s for the humanized LIV-1 mAb HBLB and HBLB S239C were alsodetermined for cyno LIV-1 expressed as a recombinant protein in a CHOcell line. Both antibodies bound with equal affinity (better than murineLIV-1 mAb).

Expression Data for LIV-1

Murine LIV-1 mAbs (at least 2 for concordance) were used forimmunohistochemical analysis of various tumor types using formalin-fixedparaffin embedded tissues.

TABLE 4 Summary of the expression data for LIV-1 in tumor samples OriginType LIV-1+ # cases % Breast Primary & metastatic 28-46 (TMA) Primarytumors 12 12 100 Metastatic tumors 17 19 89 Post-hormone treatment 19 2286 Triple Negative 13 20 65 Prostate Metastatic hormone 15 25 60refractory: bone mets soft tissue mets 21 25 84 Ovarian Primary (TMA) 972 13 Metastatic (TMA) 4 11 36 Post-chemo treated 5 17 29 Endometrial 756 12 Squamous cell Primary tumors 8 114 7 carcinoma (uterine andmultiple organs) Pancreatic Primary tumors 9 95 9 Lung Primary tumors 3192 2 (TMA)

We observed lower LIV-1 IHC positivity in studies done using tissuemicroarrays compared to large tissue sections. The difference inexpression is highly significant suggesting analysis of LIV-1 expressionin larger tissue sections is preferred. There was good concordance ofexpression using at least 2 different anti-LIV-1 mAbs. FIGS. 6 and 7show a high level of LIV-1 expression in post-hormone (tamoxifen oraromatase inhibitors) treated breast and prostate tumors providing astrong rationale to target these tumors using a LIV-1 ADC. FIG. 8 showsdetectable LIV-1 expression in triple negative (ER-, PgR-, Her2-) breastcancer tissues. The LIV-1 level of expression in triple negative breastcancer by immunohistochemistry staining was comparable to the level inthe PC3 animal model, where we demonstrated anti-tumor activity of LIV-1ADC. Triple negative breast cancers are therefore a potential targetpopulation, particularly triple negative breast cancers which have beenfound to express LIV-1.

In Vitro Anti-Tumor Activity of hLIV-14 mAb as ADC and Effector FunctionEnhanced mAb (SEA)

Anti-tumor activity of LIV-1 ADCs in vitro was measured using bothcytotoxicity assays (FIG. 9) and antibody dependent cell cytotoxicity(ADCC) (FIGS. 10 and 11). First, we performed a survey of LIV-1expression in various cell lines by quantitative FACS analysis. Thebreast cancer cell line MCF-7 from ATCC had the highest level of LIV-1binding sites/cell, as compared to the MCF-7 cell line from othersources (data not shown). We used this cell line for both assays invitro. Referring to FIG. 9, various hLIV-14 ADCs (the HBLB antibodyconjugated with vcMMAE (referred to as 1006) or mcMMAF (referred to as1269) (both small molecules and/or linkers described in US20050238649))were highly effective in killing MCF-7 cells, as compared with thenonbinding and murine control conjugates (mIgG-1006, mIgG-1269,hIgG-1006 and hIgG-1269). In addition, cysteine mutant LIV-14d ADCs,having an average of two drug linkers per antibody were also highlyeffective in killing MCF-7 cells as measured by the cytotoxic assay.Referring to FIGS. 10 and 11, in ADCC assays the activity of thefucosylated/wild-type (WT) mAb and ADCs were compared with theeffector-function enhanced versions (non-fucosylated mAbs and ADCs,referred to as SEA). The results demonstrated that effector functionenhanced LIV-1 mAbs and ADCs have good ADCC activity against MCF-7cells, as compared to non-effector function enhanced mAbs or ADCs(compare, for example, FIG. 10 hLIV-1 SEA vcMMAE with hLIV-1 vcMMAE).Referring again to FIG. 9, an effector function enhanced LIV-1 ADC(indicated as SEA) also had a similar level of cytotoxic activity aswildtype (non-fucosylated) ADCs (compare hLIV-1 SEA 1006 (vcMMAE) withhLIV-1 1006 (vcMMAE)). Thus cytotoxicity can be affected by botheffector function and conjugate action.

In Vivo Anti-Tumor Activity of hLIV-14 ADC

Using breast cancer (MCF-7) and prostate cancer (PC-3) models, wedetermined the anti-tumor activity of LIV-1 ADCs (chimeric and humanized(HBLB) mAbs with an average of 4 drugs per antibody) in vivo (FIGS.12-15). LIV-1 ADCs conjugated to vcMMAE showed significant tumor delaycompared to untreated and control ADCs. At least one complete regression(CR) was observed in all the studies using LIV-1-vcMMAE at 3 mg/kg witha number of animals having tumors that were static or grew slowlycompared to controls. Referring to FIG. 12, a chimeric form of theparental murine antibody conjugated to vcMMAE resulted in completeregressions in 3 out of 7 mice. Referring to FIG. 13, the same chimericADC produced a complete regression in 1 out of 8 mice. Referring to FIG.14, a humanized ADC (HBLB) conjugated to vcMMAE (hLIV-14-vcMMAE(4))produced a complete regression in 1 out of 8 mice. In addition, acysteine mutant form of the HBLB antibody, a vcMMAE drug linkerconjugated to each heavy chain at position 239, producing a conjugatewith an average drug load of 2 drug linkers per antibody; designatedhLIV-14d-vcMMAE(2)) exhibited similar activity as the 4-loaded form.Referring to FIG. 15, the humanized ADC (HBLB) conjugated to vcMMAE(hLIV-14-vcMMAE(4)) produced a complete regression in 1 out of 8 mice ina prostate carcinoma model. In contrast, the activity of the two loadedcysteine mutants was not as pronounced in this model (comparehLIV-14-vcMMAE(4) with hLIV-14d-vcMMAE(2), and hLIV-14-mcMMAF(4) withhLIV-14d-mcMMAF(2). In summary, these studies demonstrate that LIV-1 ADCcan stop or delay growth of LIV-1 expressing cancers including breastand prostate.

II. Humanization of BR2-22a

BR2-22a, sometimes also referred to as mAb2, is a mouse monoclonalantibody of isotype IgG1 Kappa.

Methodologies:

Unless otherwise stated below, methods described for humanization andtesting of BR2-14a are also applicable to BR2-22.

Saturation Binding Assays

1×10⁵ antigen expressing cells (either MCF7 cells expressing humanLIV-1, 293F cells, a transfected CHO cell line expressing human LIV-1 ora transfected CHO cell line expressing cyno LIV-1) were aliquoted perwell of a 96-well v-bottom plates. AlexaFluor-647 labeled murine BR2-22awas added in concentrations ranging from 0.66 pM to 690 nM and incubatedon ice for 30 minutes. Cells were pelleted and washed 3× with PBS/BSA.The cells were then pelleted and resuspended in 125 μL of PBS/BSA.Fluorescence was analyzed by flow cytometery, using percent of saturatedfluorescent signal to determine percent bound and to subsequentlycalculate apparent Kd.

Competition Binding Assays

1×10⁵ CHO cells expressing recombinant LIV-1 in PBS were aliquoted intoeach well of a 96-well v-bottom plates on ice. The cells were incubatedfor 1 hour with 5 nM AlexaFluor-647 (AF) labeled parental BR2-22a andincreasing concentrations (from 0.038 nM to 600 nM) of unlabeledhumanized BR2-22a antibody in all combinations of humanized light ChainsLA-LG and humanized heavy chains HA-HG. Cells were pelleted and washed 3times with PBS. The cells were then pelleted and resuspended in 125 μLof PBS/BSA. Fluorescence was analyzed by flow cytometery, using percentof saturated fluorescent signal to determine percent labeled humanizedBR2-22a antibody bound and to subsequently extrapolate the EC50 byfitting the data to a sigmoidal dose-response curve with variable slope.

In Vivo Activity Study

Nude (nu/nu) mice (7-8 animals/group) were implanted with tumor cellsgrown in culture: MCF-7 (NCI) at 5×10⁶ in 25% matrigel, PC3 from ATCC(2.5×10⁶ cells in 25% matrigel), and PC3 from DSMZ (5×10⁵ in 25%matrigel). For in vivo growth of MCF-7 cells, female mice also receivedestrogen supplementation by implanting a slow-release estrogen pellet(90 day release). Dosing with either chimeric or humanized LIV-1 ADC ornonbinding control ADC (3 mg/kg) started when tumors reached 100 mm³(q4d×4 intraperitoneal injections). Tumor volumes were monitored usingcalipers and animals were euthanized when tumor volume reached ˜800 mm³.Median tumor volume plots were continued for each group until one ormore animals were euthanized. All animal procedures were performed undera protocol approved by the Institutional Animal Care and Use Committeein a facility accredited by the Association for Assessment andAccreditation of Laboratory Animal Care.

Summary of Results and Discussion

Saturation Binding

BR2-22a shows 94% identity to BR2-14a in the mature heavy chain variableregion and 91% identity in the mature light chain variable region. TheKD for the murine Liv1 of BR2-22a (Table 5) was determined for humanLIV-1 expressed as an endogenous protein in a human breast cancer cellline, in 293F cells or as a recombinant protein in a CHO cell line. TheKD for BR2-22a was also determined for cyno LIV-1 expressed as arecombinant protein in a CHO cell line.

TABLE 5 Affinity measurements of BR2-22a for human (hLIV-1) and cynoLIV-1 (cyLIV-1). Cell line Antigen Kd (nM) MCF7 (ATCC) hLIV-1 1.1 293F(hLIV-1) hLIV-1 0.5 Cho hLIV-1 hLIV-1 1.5 Cho cyLIV-1 cLIV-1 4.2Humanization Strategy

The BR2-22a antibody was humanized using a VH1-02 JH5 germline acceptorsequence for the heavy chain and a VK2-30 JK4 acceptor sequence for thelight chain. These acceptor sequences were chosen based on their havingthe highest sequence identity to the mature variable region frameworksof BR2-22A heavy and light chains. Initially five variant heavy chainswere constructed. Each included the three Kabat CDRs from the heavychain of BR2-22a, the chains differing in having from zero (VA) to 11(VE) backmutations. Initially six variant light chains were constructed.Each included the three Kabat CDRs from the light chain of BR2-22a andfrom zero (LA) to four backmutations (LF). These backmutations werechosen as a result of modeling the BR2-22A antibody to identifypositions with potential to interact with antigen directly, affect CDRconformation or affect the interface between heavy and light chains andbased on prior experience in humanizing BR2-14a because of the highsequence identity between BR2-14a and BR2-22a. In fact, the same elevenpositions in the heavy chain and same four positions in the light chainwere considered for backmutation in both BR2-14a and BR2-22a (L39 wasnot considered in BR2-22a because the mouse residue is the same as thehuman residue). The back mutations present in each variant of humanizedBR2-22a are shown in Tables 6 and 7 below.

TABLE 6 V_(H) VH exon acceptor variant sequence donor framework residueshV_(H) A VH1-02 None hV_(H) B VH1-02 H29, H30, H76 hV_(H) C VH1-02 H66,H67, H71 hV_(H) D VH1-02 H27, H93, H94 hV_(H) E VH1-02 H27, H28, H29,H30, H48, H66, H67, H71, H76, H93, H94 hV_(H) F VH1-02 H27, H29, H30,H94 hV_(H) G VH1-02 H27, H29, H30, H76, H94

TABLE 7 V_(L) VL exon acceptor variant sequence donor framework residueshV_(K) A VK2-30 None hV_(K) B VK2-30 L36 hV_(K) C VK2-30 L37 hV_(K) DVK2-30 L45 hV_(K) E VK2-30 L46 hV_(K) F VK2-30 L36, L37, L45, L46 hV_(K)G VK2-30 L36, L46

The full sequence of the mature variable region of each variant is shownin FIGS. 16A and 16B.

All permutations of these five heavy chains and six light chains werethen tested in a competition assay compared with BR2-22a (see FIG. 17).Surprisingly, in view of the experience with the BR2-14a antibody inwhich improved binding relative to the mouse antibody was obtained withonly four backmutations and further backmutations did not necessarilyimprove binding affinity, the only combination of humanized chains thatshowed binding affinity approximating that of BR2-22a was HELF with 15backmutations. The other permutations showed poor or no significantbinding to LIV-1. The EC50s of the different permutations are shown inTable 8 below.

TABLE 8 EC50s for humanized BR2-22a antibodies EC50 Ab (μg/mL) HALA DNBHALB DNB HALC DNB HALD DNB HALE DNB HALF 33.2  HBLA DNB HBLB 4.9 HBLCDNB HELD DNB HBLE DNB HBLF 6.5 HCLA DNB HCLB >100    HCLC DNB HCLD DNBHCLE DNB HCLF >100    HDLA DNB HDLB DNB HDLC DNB HDLD DNB HDLE DNB HDLF14.4  HELA 68.2  HELB >100    HELC 65.7  HELD >100    HELE 25.1  HELF0.3 HELG 0.2 HFLF 0.8 HFLG 0.8 HGLF 0.4 HGLG 0.5 DNB means did not bind

Although HELF shows satisfactory binding, the antibody contains a totalof 15 backmutations, a number larger than ideal with respect topotential immunogenicity. Therefore, the HE and LF chains weresystematically varied to test the effect of removing individualbackmutations. FIG. 18 shows the variants tested. LF-1 through LF-4differ from LF in each lacking a different backmutation present in LF.Similarly, HE-1 through HE-11 lack one of the backmutations present inHE. FIG. 19 compares LF-1 through LF-4 (each paired with HE). FIG. 19shows that LF-2 and LF-3 lose substantial binding affinity relative toLF (indicated as HELF historic control in the graph), whereas LF-1 andLF-4 do not. It is concluded that backmutations L36 and L46 contributesubstantially to retention of binding affinity, while backmutations atpositions L37 and L45 can be dispensed without significant effect onbinding. FIG. 20 shows similar binding curves for the HE variants. FIG.20 shows that HE-11 lost most of its binding indicating thatbackmutation at position H94 has the greatest effect on binding affinityof the backmutations tested. Loss of backmutations at positions H27, H29and H30 also caused significant loss of affinity. The role of H30 can berationalized by the mouse residue being the result of somatic mutation.Loss of a back mutation at position H76 caused some loss of affinity.The other back mutations at positions H28, H48, H66, H67, H71 and H93could be dispensed with little or no effect on binding affinity.

In light of these experiments, heavy chains HF and HG were constructedas was light chain LG. HF included backmutations at H27, H29, H30 andH94 and HG included these mutations and a backmutation at H76. LGcontains backmutations at L36 and L46. Several permutations of HF, HG,LE and LF were tested for competition binding as shown in FIG. 21 andall showed binding within a factor of three of that of mouse BR2-22a.

In light of this experiment, HGLG was selected for further experimentsas representing the best combination of binding affinity and fewestbackmutations. This antibody is hereafter referred to as hLIV22. Thesaturation binding affinity of hLIV22 for human and cyno LIV-1 expressedfrom CHO cells is shown in FIG. 22 compared with that of hLIV14. FIG. 22shows that hLIV22 has about four fold higher affinity (inverse ofdissociation constant) for human LIV-1 than does hLIV14. Furthermore,the affinity of hLIV22 for human LIV-1 is the same within experimentalerror as its affinity for cynomolgus LIV-1, whereas hLIV14 shows twicethe affinity for human LIV-1 as for cynomolgus LIV-1. The affinity ofhLIV22 for human LIV-1 is the same within experimental error as that ofthe parent mouse antibody, BR2-22a.

In Vitro Anti-Tumor Activity of hLIV22 ADCs

Anti-tumor activity of hLIV22 ADC in vitro was measured usingcytotoxicity assays. First, we performed a survey of LIV-1 expression invarious cell lines by quantitative FACS analysis. The breast cancer cellline MCF-7 from ATCC had the highest level of LIV-1 binding sites/cell,as compared to the MCF-7 cell line from other sources (data not shown).We used this cell line for in vitro assays. We observed that varioushLIV22 ADCs (conjugated with vcMMAE (referred to as 1006) or mcMMAF(referred to as 1269) (both small molecules described in US2005-0238649)) were highly effective in killing MCF-7 cells as measuredby the in vitro cytotoxic assay. FIGS. 23 and 24 comparehLIV22-conjugated to 1006 or 1269 with a nonbinding control antibodyconjugated to 1006 or 1269.

In Vivo Anti-Tumor Activity of LIV-1 ADC

Using prostate cancer (PC-3) and breast cancer (MCF-7) models as shownin FIGS. 25 and 26, we determined the anti-tumor activity of hLIV22 ADCs(with an average of 4 drugs per antibody) in vivo. hLIV22 ADCsconjugated to vcMMAE showed significant tumor delay compared tountreated and control ADCs. There were multiple complete regressions wasobserved in the MCF-7 study using hLIV22-vcMMAE at 3 mg/kg.Additionally, in all studies there were a number of animals that hadtumors that were static or grew slowly compared to controls. Thesestudies demonstrate that hLIV22 ADC can stop or delay growth of LIV-1expressing cancers, including breast and prostate. FIG. 27 compares theactivity of hLIV22 and hLIV14 ADCs in the MCF-7 model. Although bothantibodies were effective, hLIV22 was slightly more effective. hLIV22ADCs were also tested in a model of cervical cancer. A HeLA cellxenograft model was used for the assay. After tumors grew to anappropriate size, hLIV22 conjugated to vcMMAE was administered toanimals at 3 mg/kg and 1 mg/kg. A control antibody conjugate wasadministered at 3 mg/kg. Complete and partial regression were observedin animals that received 3 mg/kg hLIV22 vc MMAE conjugate. (Data notshown.) Thus, LIV-1 antibodies and antibody drug conjugates can be usedto treat LIV-1 expressing cervical cancers.

III. Treatment of Skin Cancer Using Anti-LIV-1 Antibodies

Expression of LIV-1 Protein on Melanoma Tumor Samples

Melanoma samples from patients were assessed for LIV-1 expression, usingIHC staining. FFPE slides were de-paraffinized using Bond™ Dewaxsolution (Leica, cat # AR9222) at 72° C. Antigen retrieval was performedusing EDTA based Bond™ Epitope Retrieval Solution 2 (Leica, cat #AR9640) for 20 min at 100° C. For IHC staining we used alkalinephosphatase based detection kit: Bond™ Polymer Refine Red Detection kit(Leica, cat # DS9390). Slides were incubated with murine monoclonalprimary antibodies against LIV-1 (BR2-14a) for 45 min at 1 μg/ml withpreliminary 30 min protein block (DAKO cat #X0909). Mouse IgG (Sigma,cat # M5284) was used as negative control. After chromogen development,sections were counterstained with hematoxylin and coverslipped. Slideswere evaluated and scored by pathologist.

Results are shown in FIG. 28. Seventy-two percent of the tested melanomapatient samples (21/29) were positive for LIV-1 expression. Thisindicates that LIV-1 inhibitors, e.g., anti-LIV-1 antibodies, can beused to treat melanoma cancers.

In Vivo Anti-Melanoma Activity of LIV-1 ADC

Nude (nu/nu) mice (7-8 animals/group) are implanted with 10×10⁶ SK-MEL-5cells (a melanoma tumor-derived cell line) grown in culture. Tumors areallowed to grow in vivo until they are 100 mm³, as measured using acaliper. Humanized LIV-1 ADCs, e.g., hLIV14 or hLIV22, are administeredat 3 mg/kg. Drug conjugates are, e.g., vcMMAE or mcMMAF. Control ADC'sare also administered to control animals at 3 mg/kg. ADC's are given asq4d×4 intraperitoneal injections. Tumor volumes are monitored usingcalipers and animals are euthanized when tumor volume reaches ˜800 mm³.Administration of hLIV14 ADC or hLIV22 ADC greatly reduces tumor growthin animals as compared to those animals that received control ADC's.

Sequence listing <LIV-1 mAb light chain leader; PRT/1; mus musculus>SEQ ID NO: 1 MKLPVRLLVLMFWIPVSTS<LIV-1 mAb heavy chain leader; PRT/1; mus musculus> SEQ ID NO: 2MKCSWVIFFLMAVVLGINS <replacement heavy chain leader sequence; PRT/1;mus musculus> SEQ ID NO: 3 MAWVWTLLFLMAAAQSAQA<Light chain constant region; PRT/1; homo sapiens> SEQ ID NO: 4TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC<CH1-CH3; PRT/1; homo sapiens> SEQ ID NO: 5ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK <heavy chain CH1-CH3 (no c-term K); PRT/1;homo sapiens> SEQ ID NO: 6ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG <S239C heavy chain CH1-CH3; PRT/1; homo sapiens>SEQ ID NO: 7 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK<S239C heavy chain CH1-CH3 (no c-term K); PRT/1; homo sapiens>SEQ ID NO: 8 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG <hLIV-1 mAb HA; PRT/1; artificial> SEQ ID NO: 9QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGWIDPENGDTEYAPTFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARHDAH YGTWFAYWGQGTLVTVSS<hLIV-1 mAb HB; PRT/1; artificial> SEQ ID NO: 10QVQLVQSGAEVKKPGASVKVSCKASGYTIEDYYMHWVRQAPGQGLEWMGWIDPENGDTEYAPTFQGRVTMTRDTSINTAYMELSRLRSDDTAVYYCARHDAH YGTWFAYWGQGTLVTVSS<hLIV-1 mAb HC; PRT/1; artificial> SEQ ID NO: 11QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGWIDPENGDTEYAPTFQGKATMTADTSISTAYMELSRLRSDDTAVYYCARHDAH YGTWFAYWGQGTLVTVSS<hLIV-1 mAb HD; PRT/1; artificial> SEQ ID NO: 12QVQLVQSGAEVKKPGASVKVSCKASGFTFTDYYMHWVRQAPGQGLEWMGWIDPENGDTEYAPTFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCNVHDAH YGTWFAYWGQGTLVTVSS<hLIV-1 mAb HE; PRT/1; artificial> SEQ ID NO: 13QVQLVQSGAEVKKPGASVKVSCKASGFNIEDYYMHWVRQAPGQGLEWIGWIDPENGDTEYAPTFQGKATMTADTSINTAYMELSRLRSDDTAVYYCNVHDAH YGTWFAYWGQGTLVTVSS<hLIV-1 mAb LA; PRT/1; artificial> SEQ ID NO: 14DVVMTQSPLSLPVTLGQPASISCRSSQSIIRNDGNTYLEWFQQRPGQSPRRLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYT FGGGTKVEIKR<hLIV-1 mAb LB; PRT/1; artificial> SEQ ID NO: 15DVVMTQSPLSLPVTLGQPASISCRSSQSIIRNDGNTYLEWYQQRPGQSPRRLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYT FGGGTKVEIKR<hLIV-1 mAb LC; PRT/1; artificial> SEQ ID NO: 16DVVMTQSPLSLPVTLGQPASISCRSSQSIIRNDGNTYLEWFLQRPGQSPRRLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYT FGGGTKVEIKR<hLIV-1 mAb LD; PRT/1; artificial> SEQ ID NO: 17DVVMTQSPLSLPVTLGQPASISCRSSQSIIRNDGNTYLEWFQQRPGQSPKRLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYT FGGGTKVEIKR<hLIV-1 mAb LE; PRT/1; artificial> SEQ ID NO: 18DVVMTQSPLSLPVTLGQPASISCRSSQSIIRNDGNTYLEWFQQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYT FGGGTKVEIKR<hLIV-1 mAb LF; PRT/1; artificial> SEQ ID NO: 19DVVMTQSPLSLPVTLGQPASISCRSSQSIIRNDGNTYLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYT FGGGTKVEIKRDNA sequences: <LIV-1 mAb heavy chain leader; DNA; mus musculus>SEQ ID NO: 20 atgaaatgcagctgggtcatcttcttcctgatggcagtggttctaggaatc aattca<LIV-1 mAb light chain leader; DNA; mus musculus> SEQ ID NO: 21atgaagttgcctgttaggctgttggtgctgatgttctggattcctgtttct accagt<replacement heavy chain leader sequence; DNA; mus musculus>SEQ ID NO: 22 atggcttgggtgtggaccttgctattcctgatggcagctgcccaaagtgcc caagca<light chain constant region; DNA; mus musculus> SEQ ID NO: 23acggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaac aggggagagtgt<CH1-CH3; DNA; homo sapiens> SEQ ID NO: 24gctagcaccaagggcccatctgtcttccccctggcaccctcctccaagagcacctctgggggcacagctgccctgggctgcctggtcaaggactacttccctgaacctgtgacagtgtcctggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctccgggtaaa <CH1-CH3 (w/o c-term K); DNA; homo sapiens>SEQ ID NO: 25 gctagcaccaagggcccatctgtcttccccctggcaccctcctccaagagcacctctgggggcacagctgccctgggctgcctggtcaaggactacttccctgaacctgtgacagtgtcctggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagc ctctccctgtctccgggt<S239C CH1-CH3; DNA; artificial> SEQ ID NO: 26gctagcaccaagggcccatctgtcttccccctggcaccctcctccaagagcacctctgggggcacagctgccctgggctgcctggtcaaggactacttccctgaacctgtgacagtgtcctggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtgtgtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctccgggtaaa <S239C CH1-CH3 (w/o c-term K); DNA; artificial>SEQ ID NO: 27 gctagcaccaagggcccatctgtcttccccctggcaccctcctccaagagcacctctgggggcacagctgccctgggctgcctggtcaaggactacttccctgaacctgtgacagtgtcctggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtgtgtcttcctcttccccccaaaacccaaggacaccctcatgatetcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagc ctctccctgtctccgggt<hLTV-1 mAb HA; DNA; artificial> SEQ ID NO: 28caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggatacaccttcacagactactatatgcactgggtgaggcaggcccctggacaagggcttgagtggatgggatggattgatcctgagaatggtgatactgaatatgcccccaccttccagggcagggtcaccatgaccagggacacctccatcagcacagcctacatggagctgagcaggctgagatctgatgacacagctgtgtattactgtgccagacatgatgctcactatgggacctggtttgcttactggggccaaggaaccctggtcacagtctcc tca<hLIV-1 mAb HB; DNA; artificial> SEQ ID NO: 29caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggatacaccattgaagactactatatgcactgggtgaggcaggcccctggacaagggcttgagtggatgggatggattgatcctgagaatggtgatactgaatatgcccccaccttccagggcagggtcaccatgaccagggacacctccatcaacacagcctacatggagctgagcaggctgagatctgatgacacagctgtgtattactgtgccagacatgatgctcactatgggacctggtttgcttactggggccaaggaaccctggtcacagtctcc tca<hLIV-1 mAb HC; DNA; artificial> SEQ ID NO: 30caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggccteagtgaaggtctcctgcaaggcttctggatacaccttcacagactactatatgcactgggtgaggcaggcccctggacaagggcttgagtggatgggatggattgatcctgagaatggtgatactgaatatgcccccaccttccagggcaaggccactatgactgcagacacctccatcagcacagcctacatggagctgagcaggctgagatctgatgacacagctgtgtattactgtgccagacatgatgctcactatgggacctggtttgcttactggggccaaggaaccctggtcacagtctcc tca<hLIV-1 mAb HD; DNA; artificial> SEQ ID NO: 31caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggattcaccttcacagactactatatgcactgggtgaggcaggcccctggacaagggcttgagtggatgggatggattgatcctgagaatggtgatactgaatatgcccccaccttccagggcagggtcaccatgaccagggacacctccatcagcacagcctacatggagctgagcaggctgagatctgatgacacagctgtgtattactgtgccagacatgatgctcactatgggacctggtttgcttactggggccaaggaaccctggtcacagtctcc tca<hLIV-1 mAb HE; DNA; artificial> SEQ ID NO: 32 Caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggattcaacattgaagactactatatgcactgggtgaggcaggcccctggacaagggcttgagtggattggatggattgatcctgagaatggtgatactgaatatgcccccaccttccagggcaaggccactatgactgcagacacctccatcaacacagcctacatggagctgagcaggctgagatctgatgacacagctgtgtattactgtaatgtccatgatgctcactatgggacctggtttgcttactggggccaaggaaccctggtcacagtctcc tca<hLIV-1 mAb LA; DNA; artificial> SEQ ID NO: 33gatgttgtgatgactcagtctccactctccctgcctgtcacccttggacagcctgcctccatctcctgcagatctagtcagagcattataaggaatgatggaaacacctatttggaatggtttcagcagaggccaggccaatctccaaggaggctaatttatagagtttccaacaggttttctggggtcccagacagattctctggcagtgggtcaggcactgatttcacactgaaaatcagcagggtggaggctgaggatgttggggtttattactgctttcaaggttcacatgttccctacacctttggaggagggaccaaggtggagatcaaacgt <hLIV-1 mAb LB; DNA; artificial>SEQ ID NO: 34 gatgttgtgatgactcagtctccactctccctgcctgtcacccttggacagcctgcctccatctcctgcagatctagtcagagcattataaggaatgatggaaacacctatttggaatggtaccagcagaggccaggccaatctccaaggaggctaatttatagagtttccaacaggttttctggggtcccagacagattctctggcagtgggtcaggcactgatttcacactgaaaatcagcagggtggaggctgaggatgttggggtttattactgctttcaaggttcacatgttccctacacctttggaggagggaccaaggtggagatcaaacgt <hLIV-1 mAb LC; DNA; artificial>SEQ ID NO: 35 gatgttgtgatgactcagtctccactctccctgcctgtcacccttggacagcctgcctccatctcctgcagatctagtcagagcattataaggaatgatggaaacacctatttggaatggtttctgcagaggccaggccaatctccaaggaggctaatttatagagtttccaacaggttttctggggtcccagacagattctctggcagtgggtcaggcactgatttcacactgaaaatcagcagggtggaggctgaggatgttggggtttattactgctttcaaggttcacatgttccctacacctttggaggagggaccaaggtggagatcaaacgt <hLIV-1 mAb LD; DNA; artificial>SEQ ID NO: 36 gatgttgtgatgactcagtctccactctccctgcctgtcacccttggacagcctgcctccatctcctgcagatctagtcagagcattataaggaatgatggaaacacctatttggaatggtttcagcagaggccaggccaatctccaaagaggctaatttatagagtttccaacaggttttctggggtcccagacagattctctggcagtgggtcaggcactgatttcacactgaaaatcagcagggtggaggctgaggatgttggggtttattactgctttcaaggttcacatgttccctacacctttggaggagggaccaaggtggagatcaaacgt <hLTV-1 mAb LE; DNA; artificial>SEQ ID NO: 37 gatgttgtgatgactcagtctccactctccctgcctgtcacccttggacagcctgcctccatctcctgcagatctagtcagagcattataaggaatgatggaaacacctatttggaatggtttcagcagaggccaggccaatctccaaggctcctaatttatagagtttccaacaggttttctggggtcccagacagattctctggcagtgggtcaggcactgatttcacactgaaaatcagcagggtggaggctgaggatgttggggtttattactgctttcaaggttcacatgttccctacacctttggaggagggaccaaggtggagatcaaacgt <hLIV-1 mAb LF; DNA; artificial>SEQ ID NO: 38 gatgttgtgatgactcagtctccactctccctgcctgtcacccttggacagcctgcctccatctcctgcagatctagtcagagcattataaggaatgatggaaacacctatttggaatggtacctgcagaaaccaggccaatctccaaagctcctaatttatagagtttccaacaggttttctggggtcccagacagattctctggcagtgggtcaggcactgatttcacactgaaaatcagcagggtggaggctgaggatgttggggtttattactgctttcaaggttcacatgttccctacacctttggaggagggaccaaggtggagatcaaacgt<Liv1 mAb2 light chain leader; PRT/1; mus musculus> SEQ ID NO: 39MKLPVRLLVLMFWIPVATSS <Liv1 mAb2 heavy chain leader; PRT/1; mus musculus>SEQ ID NO: 40 MKCSWVIFFLMAVVIGINS<replacement heavy chain leader sequence; PRT/1; mus musculus>SEQ ID NO: 41 MAWVWTLLFLMAAAQSAQA<Light chain constant region; PRT/1; homo sapiens> SEQ ID NO: 42TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC<CH1-CH3; PRT/1; homo sapiens> SEQ ID NO: 43 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* <heavy chain CH1-CH3 (no c-term K); PRT/1;homo sapiens> SEQ ID NO: 44ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG <S239C heavy chain CH1-CH3; PRT/1; homo sapiens>SEQ ID NO: 45 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK<S239C heavy chain CH1-CH3 (no c-term K); PRT/1; homo sapiens>SEQ ID NO: 46 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG <hLiv1 mAb2 HA; PRT/1; artificial> SEQ ID NO: 47QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGWIDPENGDTEYGPKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARHNAH YGTWFAYWGQGTLVTVSS<hLiv1 mAb2 HB; PRT/1; artificial> SEQ ID NO: 48QVQLVQSGAEVKKPGASVKVSCKASGYTIEDYYMHWVRQAPGQGLEWMGWIDPENGDTEYGPKFQGRVTMTRDTSINTAYMELSRLRSDDTAVYYCARHNAH YGTWFAYWGQGTLVTVSS<hLiv1 mAb2 HC; PRT/1; artificial> SEQ ID NO: 49QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGWIDPENGDTEYGPKFQGKATMTADTSISTAYMELSRLRSDDTAVYYCARHNAH YGTWFAYWGQGTLVTVSS<hLiv1 mAb2 HD; PRT/1; artificial> SEQ ID NO: 50QVQLVQSGAEVKKPGASVKVSCKASGLTFTDYYMHWVRQAPGQGLEWMGWIDPENGDTEYGPKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCTVHNAH YGTWFAYWGQGTLVTVSS<hLiv1 mAb2 HE; PRT/1; artificial> SEQ ID NO: 51QVQLVQSGAEVKKPGASVKVSCKASGLNIEDYYMHWVRQAPGQGLEWIGWIDPENGDTEYGPKFQGKATMTADTSINTAYMELSRLRSDDTAVYYCTVHNAH YGTWFAYWGQGTLVTVSS<hLiv1 mAb2 HF; PRT/1; artificial> SEQ ID NO: 52QVQLVQSGAEVKKPGASVKVSCKASGLTIEDYYMHWVRQAPGQGLEWMGWIDPENGDTEYGPKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAVHNAH YGTWFAYWGQGTLVTVSS<hLiv1 mAb2 HG; PRT/1; artificial> SEQ ID NO: 53QVQLVQSGAEVKKPGASVKVSCKASGLTIEDYYMHWVRQAPGQGLEWMGWIDPENGDTEYGPKFQGRVTMTRDTSINTAYMELSRLRSDDTAVYYCAVHNAH YGTWFAYWGQGTLVTVSS<hLiv1 mAb2 LA; PRT/1; artificial> SEQ ID NO: 54DVVMTQSPLSLPVTLGQPASISCRSSQSLLHSSGNTYLEWFQQRPGQSPRRLIYKISTRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYT FGGGTKVEIKR<hLiv1 mAb2 LB; PRT/1; artificial> SEQ ID NO: 55DVVMTQSPLSLPVTLGQPASISCRSSQSLLHSSGNTYLEWYQQRPGQSPRRLIYKISTRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYT FGGGTKVEIKR<hLiv1 mAb2 LC; PRT/1; artificial> SEQ ID NO: 56DVVMTQSPLSLPVTLGQPASISCRSSQSLLHSSGNTYLEWFLQRPGQSPRRLIYKISTRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYT FGGGTKVEIKR<hLiv1 mAb2 LD; PRT/1; artificial> SEQ ID NO: 57DVVMTQSPLSLPVTLGQPASISCRSSQSLLHSSGNTYLEWFQQRPGQSPKRLIYKISTRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYT FGGGTKVEIKR<hLiv1 mAb2 LE; PRT/1; artificial> SEQ ID NO: 58DVVMTQSPLSLPVTLGQPASISCRSSQSLLHSSGNTYLEWFQQRPGQSPRPLIYKISTRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYT FGGGTKVEIKR<hLiv1 mAb2 LF; PRT/1; artificial> SEQ ID NO: 59DVVMTQSPLSLPVTLGQPASISCRSSQSLLHSSGNTYLEWYLQRPGQSPKPLIYKISTRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYT FGGGTKVEIKR<hLiv1 mAb2 LG; PRT/1; artificial> SEQ ID NO: 60DVVMTQSPLSLPVTLGQPASISCRSSQSLLHSSGNTYLEWYQQRPGQSPRPLIYKISTRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYT FGGGTKVEIKRDNA sequences: <Liv1 mAb2 heavy chain leader; DNA; mus musculus>SEQ ID NO: 61 atgaaatgcagctgggtcatcttcttcctgatggcagtggttataggaatc aattca<Liv1 mAb2 light chain leader; DNA; mus musculus> SEQ ID NO: 62atgaagttgcctgttaggctgttggtgctgatgttctggattcctgctacc agcagt<replacement heavy chain leader sequence; DNA; mus musculus>SEQ ID NO: 63 atggcttgggtgtggaccttgctattcctgatggcagctgcccaaagtgcc caagca<light chain constant region; DNA; homo sapiens> SEQ ID NO: 64acgacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttc aacaggggagagtgttag<CH1-CH3; DNA; homo sapiens> SEQ ID NO: 65gctagcaccaagggcccatctgtcttccccctggcaccctcctccaagagcacctctgggggcacagctgccctgggctgcctggtcaaggactacttccctgaacctgtgacagtgtcctggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctccgggtaaa <CH1-CH3 (w/o c-term K); DNA; homo sapiens>SEQ ID NO: 66 gctagcaccaagggcccatctgtcttccccctggcaccctcctccaagagcacctctgggggcacagctgccctgggctgcctggtcaaggactacttccctgaacctgtgacagtgtcctggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatetcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagc ctctccctgtctccgggt<S239C CH1-CH3; DNA; artificial> SEQ ID NO: 67gctagcaccaagggcccatctgtcttccccctggcaccctcctccaagagcacctctgggggcacagctgccctgggctgcctggtcaaggactacttccctgaacctgtgacagtgtcctggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtgtgtcttcctcttccccccaaaacccaaggacacccteatgatetcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctccgggtaaa <S239C CH1-CH3 (w/o c-term K); DNA; artificial>SEQ ID NO: 68 gctagcaccaagggcccatctgtcttccccctggcaccctcctccaagagcacctctgggggcacagctgccctgggctgcctggtcaaggactacttccctgaacctgtgacagtgtcctggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccaqcagettgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtgtgtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttcteatgetccgtgatgcatgaggctctgcacaaccactacacacagaagagc ctctccctgtctccgggt<hLiv1 mAb2 HA; DNA; artificial> SEQ ID NO: 69caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggatacaccttcacagactactatatgcactgggtgaggcaggcccctggacaagggcttgagtggatgggatggattgatcctgaaaatggtgatactgaatatggcccgaagttccagggcagggtcaccatgaccagggacacctccatcagcacagcctacatggagctgagcaggctgagatctgatgacacagctgtgtattactgtgccagacataatgctcactacgggacctggtttgcttactggggccaaggaaccctggtcacagtctcc tca<hLiv1 mAb2 HB; DNA; artificial> SEQ ID NO: 70 caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggatacaccattgaagactactatatgcactgggtgaggcaggcccctggacaagggcttgagtggatgggatggattgatcctgaaaatggtgatactgaatatggcccgaagttccagggcagggtcaccatgaccagggacacctccatcaacacagcctacatggagctgagcaggctgagatctgatgacacagctgtgtattactgtgccagacataatgctcactacgggacctggtttgcttactggggccaaggaaccctggtcacagtctcc tca<hLiv1 mAb2 HC; DNA; artificial> SEQ ID NO: 71caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggatacaccttcacagactactatatgcactgggtgaggcaggcccctggacaagggcttgagtggatgggatggattgatcctgaaaatggtgatactgaatatggcccgaagttccagggcaaggccaccatgaccgcagacacctccatcagcacagcctacatggagctgagcaggctgagatctgatgacacagctgtgtattactgtgccagacataatgctcactacgggacctggtttgcttactggggccaaggaaccctggtcacagtctcc tca<hLiv1 mAb2 HD; DNA; artificial> SEQ ID NO: 72Caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggactcaccttcacagactactatatgcactgggtgaggcaggcccctggacaagggcttgagtggatgggatggattgatcctgaaaatggtgatactgaatatggcccgaagttccagggcagggtcaccatgaccagggacacctccatcagcacagcctacatggagctgagcaggctgagatctgatgacacagctgtgtattactgtactgtccataatgctcactacgggacctggtttgcttactggggccaaggaaccctggtcacagtctcc tca<hLiv1 mAb2 HE; DNA; artificial> SEQ ID NO: 73caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggactcaacattgaagactactatatgcactgggtgaggcaggcccctggacaagggcttgagtggattggatggattgatcctgaaaatggtgatactgaatatggcccgaagttccagggcaaggccaccatgaccgcagacacctccatcaacacagcctacatggagctgagcaggctgagatctgatgacacagctgtgtattactgtactgtccataatgctcactacgggacctggtttgcttactggggccaaggaaccctggtcacagtctcc tca<hLiv1 mAb2 HF; DNA; artificial> SEQ ID NO: 74caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggactcaccattgaagactactatatgcactgggtgaggcaggcccctggacaagggcttgagtggatgggatggattgatcctgaaaatggtgatactgaatatggcccgaagttccagggcagggtcaccatgaccagggacacctccatcagcacagcctacatggagctgagcaggctgagatctgatgacacagctgtgtattactgtgccgtccataatgctcactacgggacctggtttgcttactggggccaaggaaccctggtcacagtctcc tca<hLiv1 mAb2 HG; DNA; artificial> SEQ ID NO: 75caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggactcaccattgaagactactatatgcactgggtgaggcaggcccctggacaagggcttgagtggatgggatggattgatcctgaaaatggtgatactgaatatggcccgaagttccagggcagggtcaccatgaccagggacacctccatcaacacagcctacatggagctgagcaggctgagatctgatgacacagctgtgtattactgtgccgtccataatgctcactacgggacctggtttgcttactggggccaaggaaccctggtcacagtctcc tca<hLiv1 mAb2 LA; DNA; artificial> SEQ ID NO: 76gatgttctggattcctgctaccagcagtgatgttgtgatgactcagtctccactctccctgcctgtcacccttggacagcctgcctccatctcctgcagatctagtcagagccttttacacagtagtggaaacacctatttagaatggtttcagcagaggccaggccaatctccaaggaggctaatttataaaatttccacccgattttctggggtcccagacagattctctggcagtgggtcaggcactgatttcacactgaaaatcagcagggtggaggctgaggatgttggggtttattactgctttcaaggttcacatgttccctacacctttggaggagggaccaaggtgga gatcaaacgtacg<hLiv1 mAb2 LB; DNA; artificial> SEQ ID NO: 77gatgttctggattcctgctaccagcagtgatgttgtgatgactcagtctccactctccctgcctgtcacccttggacagcctgcctccatctcctgcagatctagtcagagccttttacacagtagtggaaacacctatttagaatggtaccagcagaggccaggccaatctccaaggaggctaatttataaaatttccacccgattttctggggtcccagacagattctctggcagtgggtcaggcactgatttcacactgaaaatcagcagggtggaggctgaggatgttggggtttattactgctttcaaggttcacatgttccctacacctttggaggagggaccaaggtgga gatcaaacgtacg<hLiv1 mAb2 LC; DNA; artificial> SEQ ID NO: 78gatgttctggattcctgctaccagcagtgatgttgtgatgactcagtctccactctccctgcctgtcacccttggacagcctgcctccatctcctgcagatctagtcagagccttttacacagtagtggaaacacctatttagaatggtttctgcagaggccaggccaatctccaaggaggctaatttataaaatttccacccgattttctggggtcccagacagattctctggcagtgggtcaggcactgatttcacactgaaaatcagcagggtggaggctgaggatgttggggtttattactgctttcaaggttcacatgttccctacacctttggaggagggaccaaggtgga gatcaaacgtacg<hLiv1 mAb2 LD; DNA; artificial> SEQ ID NO: 79gatgttctggattcctgctaccagcagtgatgttgtgatgactcagtctccactctccctgcctgtcacccttggacagcctgcctccatctcctgcagatctagtcagagccttttacacagtagtggaaacacctatttagaatggtttcagcagaggccaggccaatctccaaagaggctaatttataaaatttccacccgattttctggggtcccagacagattctctggcagtgggtcaggcactgatttcacactgaaaatcagcagggtggaggctgaggatgttggggtttattactgctttcaaggttcacatgttccctacacctttggaggagggaccaaggtgga gatcaaacgtacg<hLiv1 mAb2 LE; DNA; artificial> SEQ ID NO: 80gatgttctggattcctgctaccagcagtgatgttgtgatgactcagtctccactctccctgcctgtcacccttggacagcctgcctccatctcctgcagatctagtcagagccttttacacagtagtggaaacacctatttagaatggtttcagcagaggccaggccaatctccaaggcccctaatttataaaatttccacccgattttctggggtcccagacagattctctggcagtgggtcaggcactgatttcacactgaaaatcagcagggtggaggctgaggatgttggggtttattactgctttcaaggttcacatgttccctacacctttggaggagggaccaaggtgga gatcaaacgtacg<hLiv1 mAb2 LF; DNA; artificial> SEQ ID NO: 81gatgttgtgatgactcagtctccactctccctgcctgtcacccttggacagcctgcctccatctcctgcagatctagtcagagccttttacacagtagtggaaacacctatttagaatggtacctgcagaggccaggccaatctccaaagcccctaatttataaaatttccacccgattttctggggtcccagacagattctctggcagtgggtcaggcactgatttcacactgaaaatcagcagggtggaggctgaggatgttggggtttattactgctttcaaggttcacatgttccctacacctttggaggagggaccaaggtggagatcaaacgt <hLiv1 BR2-22a LG; DNA; artificial>SEQ ID NO: 82 gatgttgtgatgactcagtctccactctccctgcctgtcacccttggacagcctgcctccatctcctgcagatctagtcagagccttttacacagtagtggaaacacctatttagaatggtaccagcagaggccaggccaatctccaaggcccctaatttataaaatttccacccgattttctggggtcccagacagattctctggcagtgggtcaggcactgatttcacactgaaaatcagcagggtggaggctgaggatgttggggtttattactgctttcaaggttcacatgttccctacacctttggaggagggaccaaggtggagatcaaacgt <Q13433; protein SEQ ID NO: 83 MARKLSVILI LTFALSVTNP LHELKAAAFP QTTEKISPNWESGINVDLAI STRQYHLQQL FYRYGENNSL SVEGFRKLLQNIGIDKIKRI HIHHDHDHHS DHEHHSDHER HSDHEHHSEHEHHSDHDHHS HHNHAASGKN KRKALCPDHD SDSSGKDPRNSQGKGAHRPE HASGRRNVKD SVSASEVTST VYNTVSEGTHFLETIETPRP GKLFPKDVSS STPPSVTSKS RVSRLAGRKTNESVSEPRKG FMYSRNTNEN PQECFNASKL LTSHGMGIQVPLNATEFNYL CPAIINQIDA RSCLIHTSEK KAEIPPKTYSLQIAWVGGFI AISIISFLSL LGVILVPLMN RVFFKFLLSFLVALAVGTLS GDAFLHLLPH SHASHHHSHS HEEPAMEMKRGPLFSHLSSQ NIEESAYFDS TWKGLTALGG LYFMFLVEHVLTLIKQFKDK KKKNQKKPEN DDDVEIKKQL SKYESQLSTNEEKVDTDDRT EGYLRADSQE PSHFDSQQPA VLEEEEVMIAHAHPQEVYNE YVPRGCKNKC HSHFHDTLGQ SDDLIHHHHDYHHILHHHHH QNHHPHSHSQ RYSREELKDA GVATLAWMVIMGDGLHNFSD GLAIGAAFTE GLSSGLSTSV AVFCHELPHELGDFAVLLKA GMTVKQAVLY NALSAMLAYL GMATGIFIGHYAENVSMWIF ALTAGLFMYV ALVDMVPEML HNDASDHGCSRWGYFFLQNA GMLLGFGIML LISIFEHKIV FRINF <AAA96258.2; proteinSEQ ID No: 84 MARKLSVILI LTFALSVTNP LHELKAAAFP QTTEKISPNWESGINVDLAI STRQYHLQQL FYRYGENNSL SVEGFRKLLQNIGIDKIKRI HIHHDHDHHS DHEHHSDHER HSDHEHHSDHEHHSDHNHAA SGKNKRKALC PDHDSDSSGK DPRNSQGKGAHRPEHASGRR NVKDSVSASE VTSTVYNTVS EGTHFLETIETPRPGKLFPK DVSSSTPPSV TSKSRVSRLA GRKTNESVSEPRKGFMYSRN TNENPQECFN ASKLLTSHGM GIQVPLNATEFNYLCPAIIN QIDARSCLIH TSEKKAEIPP KTYSLQIAWVGGFIAISIIS FLSLLGVILV PLMNRVFFKF LLSFLVALAVGTLSGDAFLH LLPHSHASHH HSHSHEEPAM EMKRGPLFSHLSSQNIEESA YFDSTWKGLT ALGGLYFMFL VEHVLTLIKQFKDKKKKNQK KPENDDDVEI KKQLSKYESQ LSTNEEKVDTDDRTEGYLRA DSQEPSHFDS QQPAVLEEEE VMIAHAHPQEVYNEYVPRGC KNKCHSHFHD TLGQSDDLIH HHHDYHHILHHHHHQNHHPH SHSQRYSREE LKDAGVATLA WMVIMGDGLHNFSDGLAIGA AFTEGLSSGL STSVAVFCHE LPHELGDFAVLLKAGMTVKQ AVLYNALSAM LAYLGMATGI FIGHYAENVSMWIFALTAGL FMYVALVDMV PEMLHNDASD HGCSRWGYFFLQNAGMLLGF GIMLLISIFE HKIVFRINF >Cyno LIV-1 SEQ ID NO: 85MARKLSVILILTFTLSVTNPLHELKSAAAFPQTTEKISPNWESGINVDLAITTRQYHLQQLFYRYGENNSLSVEGFRKLLQNIGIDKIKRIHIHHDHDHHSDHEHHSDHEHHSDHEHHSHRNHAASGKNKRKALCPEHDSDSSGKDPRNSQGKGAHRPEHANGRRNVKDSVSTSEVTSTVYNTVSEGTHFLETIETPKLFPKDVSSSTPPSVTEKSLVSRLAGRKTNESMSEPRKGFMYSRNTNENPQECFNASKLLTSHGMGIQVPLNATEFNYLCPAIINQIDARSCLIHTSEKKAEIPPKTYSLQIAWVGGFIAISIISFLSLLGVILVPLMNRVFFKFLLSFLVALAVGTLSGDAFLHLLPHSHASHHHSHSHEEPAMEMKRGPLFSHLSSQNIEESAYFDSTWKGLTALGGLYFMFLVEHVLTLIKQFKDKKKKNQKKPENDDDVEIKKQLSKYESQLSTNEEKVDTDDRTEGYLRADSQEPSHFDSQQPAILEEEEVMIAHAHPQEVYNEYVPRGCKNKCHSHFHDTLGQSDDLIHHHHDYHHILHHHHHQNHHPHSHSQRYSREELKDAGIATLAWMVIMGDGLHNFSDGLAIGAAFTEGLSSGLSTSVAVFCHELPHELGDFAVLLKAGMTVKQAVLYNALSAMLAYLGMATGIFIGHYAENVSMWIFALTAGLFMYVALVDMVPEMLHNDASDHGCSRWGYFFLQNAGMLLGFGIMLLISIFEHKIVFRINF

What is claimed is:
 1. A humanized antibody that specifically bindshuman LIV-1 comprising a mature heavy chain variable region comprisingthree CDRs of SEQ ID NO:10 and having an amino acid sequence at least90% identical to HB (SEQ ID NO:10) and a mature light chain variableregion comprising three CDRs of SEQ ID NO:15 and having an amino acidsequence at least 90% identical to LB (SEQ ID NO:15) provided thatposition H29, H30 and H76 are occupied by I, E and N, and L36 isoccupied by Y.
 2. The humanized antibody of claim 1, comprising a matureheavy chain variable region having an amino acid sequence at least 95%identical to HB and a mature light chain variable region at least 95%identical to LB.
 3. The humanized antibody of claim 1, provided that anydifference in the variable region frameworks of the mature heavy chainvariable region and SEQ ID NO:10 are selected from the group consistingof H27 occupied by F, H28 occupied by N, H48 occupied by I, H66 occupiedby K, H67 occupied by A, H71 occupied by A, H76 occupied by N, H93occupied by N, H94 occupied by V, and any differences between the maturelight chain variable regions and SEQ ID NO:15 are selected from thegroup consisting of L37 occupied by L, L39 occupied by K, L45 occupiedby K, and L46 occupied by L.
 4. The humanized antibody of claim 1,wherein the mature heavy chain variable region is fused to a heavy chainconstant region and the mature light chain variable region is fused to alight chain constant region.
 5. The humanized antibody of claim 4,wherein the heavy chain constant region is a mutant form of naturalhuman constant region which has reduced binding to an Fcgamma receptorrelative to the natural human constant region.
 6. The humanized antibodyof claim 4, wherein the heavy chain constant region is of IgG1 isotype.7. The humanized antibody of claim 4, wherein the heavy chain constantregion has an amino acid sequence comprising SEQ ID NO:6 and the lightchain constant region has an amino acid sequence comprising SEQ ID NO:4.8. The humanized antibody of claim 4, wherein the heavy chain constantregion has an amino acid sequence comprising SEQ ID NO:8 (S239C) and thelight chain constant region has an amino acid sequence comprising SEQ IDNO:4.
 9. The humanized antibody of claim 1, wherein the mature heavychain variable region has an amino acid sequence comprising SEQ ID NO:10and the mature light chain variable region has an amino acid sequencecomprising SEQ ID NO:15.
 10. The humanized antibody of claim 1, whereinthe antibody is conjugated to a cytotoxic or cytostatic agent.
 11. Ahumanized antibody that specifically binds human LIV-1 comprising amature heavy chain variable region comprising 3 CDRs of SEQ ID NO:10 andwherein positions H29, H30 and H76 are occupied by I, E and Nrespectively, and a mature light chain variable region comprising 3 CDRsof SEQ ID NO:15, and wherein position L36 is occupied by Y.
 12. Thehumanized antibody of claim 1 having greater affinity for human LIV-1than the antibody BR2-14a.
 13. The humanized antibody of claim 1, havingan association constant for human or cynomolgus monkey LIV-1 of 0.5 to2×10⁹M⁻¹.
 14. A nucleic acid encoding a mature heavy chain variableregion and/or a mature light chain variable region of claim
 1. 15. Amethod of treating a patient having a cancer expressing LIV-1,comprising administering to the patient an effective regime of ahumanized antibody of claim 1 to thereby treat the cancer expressingLIV-1.
 16. The method of claim 15, wherein the cancer is breast cancer,prostate cancer, cervical cancer, or melanoma.
 17. A pharmaceuticalcomposition comprising a humanized antibody of claim
 1. 18. The methodof claim 15, wherein the antibody is conjugated to a cytotoxic orcytostatic agent.